Abstract

Background

Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise both pre-clinically and clinically. The scFv-based constructs, including BiTE and DART, which bind monovalently to CD3 on T cells and to the target antigen on tumor cells, exhibit fast blood clearance and neurological toxicity due to their small size (∼55 kDa). Herein, we describe the generation of novel T-cell redirecting trivalent bsAbs comprising an anti-CD3 scFv covalently conjugated to a stabilized F(ab)2. The design was initially characterized with a prototype construct designated (19)-3s, which specifically targets CD19 on B cells. A panel of trivalent bsAbs was evaluated for their potential use in targeted T-cell immunotherapy of various B-cell malignancies. Potential advantages of this design include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity.

Methods

The DOCK-AND-LOCKTM (DNLTM) method was used to generate a panel of B-cell targeting bsAbs, (19)-3s, (20)-3s, (22)-3s, and (C2)-3s, which target CD19, CD20, CD22, and HLA-DR, respectively. This was achieved by combining a stabilized anti-X F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by LC-MS, SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Each construct can mediate the formation of immunological synapses between T cells and malignant B cells, resulting in T-cell activation. At an E:T ratio of 10:1, using isolated T cells as effector cells, the bsAbs induced potent T-cell-mediated cytotoxicity in various B-cell malignancies, including Burkitt lymphomas (Daudi, Ramos, Namalwa), mantle cell lymphoma (Jeko-1), and acute lymphoblastic leukemia (Nalm-6). A non-tumor binding control, (14)-3s, induced only moderate T-cell killing at >10 nM. The nature of the antigen/epitope, particularly its size and proximity to the cell surface, appears to be more important than antigen density for T-cell retargeting potency (Table 1). It is likely that (20)-3s is consistently more potent than (19)-3s and (C2)-3s, even when the expression of CD19 or HLA-DR is considerably higher than CD20, as seen with Namalwa and Jeko-1, respectively. This is likely because the CD20 epitope comprises a small extracellular loop having close proximity to the cell surface. When compared directly using Daudi, (22)-3s was the least potent. Compared to CD19 and CD20, CD22 is expressed at the lowest density, is a rapidly internalizing antigen, and its epitope is further away from the cell surface; each of these factors may contribute to its reduced potency. Finally, sensitivity to T-cell retargeted killing is cell-line-dependent, as observed using (19)-3s, where Raji (IC50 >3 nM) is largely unresponsive yet Ramos (IC50 = 2 pM) is highly sensitive, even though the former expresses higher CD19 antigen density.

Conclusions

(19)-3s, (20)-3s, (22)-3s, and (C2)-3s can bind T cells and target B cells simultaneously and induce T-cell-mediated killing in vitro. The modular nature of the DNL method allowed the rapid production of several related conjugates for redirected T-cell killing of various B-cell malignancies, without the need for additional recombinant engineering and protein production. The close proximity of the CD20 extracellular epitope to the cell surface results in the highest potency for (20)-3s, which is an attractive candidate bsAb for use in this platform. We are currently evaluating the in vivo activity of these constructs to determine if this novel bsAb format offers additional advantages.

Table 1

Ex vivo re-directed T-cell killing

  Antigen Expression2 IC503 
Cell Line Type1 CD19 CD20 CD22 HLA-DR (19)-3s (20)-3s (22)-3s (C2)-3s 
Daudi BL 1.00 1.00 1.00 1.00 0.3 N.D. 
Ramos BL 0.76 0.65 0.26 0.36 0.4 N.D. 
Nalm-6 ALL 1.63 0.05 0.19 0.17 N.D. N.D. N.D. 
Namalwa BL 0.76 0.11 0.05 0.40 63 30 >3000 N.D. 
Raji BL 1.41 0.69 0.59 0.84 >3000 N.D. N.D. N.D. 
Jeko-1 MCL 0.89 1.02 0.05 1.06 3000 N.D. 20 
  Antigen Expression2 IC503 
Cell Line Type1 CD19 CD20 CD22 HLA-DR (19)-3s (20)-3s (22)-3s (C2)-3s 
Daudi BL 1.00 1.00 1.00 1.00 0.3 N.D. 
Ramos BL 0.76 0.65 0.26 0.36 0.4 N.D. 
Nalm-6 ALL 1.63 0.05 0.19 0.17 N.D. N.D. N.D. 
Namalwa BL 0.76 0.11 0.05 0.40 63 30 >3000 N.D. 
Raji BL 1.41 0.69 0.59 0.84 >3000 N.D. N.D. N.D. 
Jeko-1 MCL 0.89 1.02 0.05 1.06 3000 N.D. 20 

1Cancer classification: BL, Burkitt lymphoma; ALL, acute lymphoblastic leukemia; MCL, mantle cell lymphoma. 2Expression level determined by flow cytometry and normalized to that of Daudi. 3IC50, the picomolar concentration that achieved 50% target cell killing.

Disclosures:

Rossi:Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other.

Author notes

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Asterisk with author names denotes non-ASH members.