Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies

Abstract

T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.

Keywords: BCMA; Bispecific antibody; CD3; T cell engager; T cells; deep sequencing; multiple myeloma; repertoire.

Figure 1.

Two different CD3 cell-binding CDRH3 sequence families were identified using NGS-based discovery followed by high-throughput recombinant expression and screening. (a) The discovery workflow combines antibody repertoire deep sequencing and custom bioinformatics analysis with high-throughput gene assembly, recombinant expression and screening. OmniFlic rats express a comprehensive human VH gene repertoire with a single pre-rearranged human kappa light chain. Endogenous rat heavy chain, kappa, and lambda loci have been knocked out. (b) Based on antibody repertoire analysis, 378 heavy chain sequences were selected for expression with a fixed human kappa light chain. All fully human antibody candidates were tested by flow cytometry for the ability to bind CD3+ Jurkat cells in a primary screen, and results are shown in a heatmap in which each row is a unique heavy chain VH sequence and the degree of red indicates cell-binding strength. The dendrogram indicates the relationships between the sequences tested in the primary screen. (c) Two cell-binding VH sequence families were identified and additional family members from the repertoire were expressed and tested for cell binding in a secondary screen. Sequence variation among family members is illustrated by colored blocks, with each color representing an amino acid residue.

Figure 2.

Members of both new anti-CD3 mAb families bind to and activate human T-cells. Two representative members were selected from each new anti-CD3 mAb family for purification and further functional assessment using flow cytometry. Antibodies were tested for the ability to bind human CD3-expressing Jurkat cells (a), and to activate primary T-cells in a sample of total human or cyno PBMCs as measured by T-cell surface expression of CD69 (b,c).

Figure 3.

A wide range of killing potency is observed among a set of bispecific antibodies using twelve novel anti-CD3 binding arms. (a) Direct comparison of anti-CD3 functional activity was enabled by combining each of 12 new anti-CD3 arms with the same anti-BCMA arm to create bispecific proteins on a silenced Fc using a standard knob-in-hole system. The BCMA binding arm was derived from a human heavy chain only antibody (CH1 deleted) that does not bind to kappa or lambda light chains. (b) BCMAxCD3 bispecific antibodies, across a broad dose spectrum, were tested for the ability to kill BCMA+ tumor cells through redirection of activated primary T-cells. Tumor cell lysis was not observed with a BCMA- control cell line (data not shown).

Figure 4.

Bispecific antibody-mediated cytokine release varies significantly between CD3_F1F- and CD3_F2B-containing bispecific antibodies. (a) Levels of specific tumor cell lysis and cytokine release for IL-2 and IFNγ were measured after resting human T-cells were cultured with NCI-H929 (BCMA-positive) or K562 (BCMA-negative) cells and increasing doses of CD3xBCMA antibodies or a negative control (CD3_F1Fx[off-target arm]). (b) The same two CD3-targeting arms were combined with an anti-CD19 arm in bispecific format, and similar primary T-cell based studies were undertaken using CD19-positive Raji cells. (c) Purified pan T-cells from 10 different healthy human donors were incubated with a BCMA+ tumor cell line (NCI-H929) in the presence of plateau killing doses for CD3_F1FxBCMA and CD3_F1Fx[off-target arm] isotype control (0.1nM) as well as CD3_F2BxBCMA and CD3_F2Bx[off-target arm] isotype control (10nM).

Figure 5.

CD3_F1F and CD3_F2B bispecific antibodies kill BCMA+ tumor cells in mice and show typical PK curves. (a) NSG mice were engrafted with Luciferase-labeled RPMI-8226 BCMA+ tumor cells, and human PBMCs were injected 6 days later. Starting four days post-PBMC addition, bispecific antibodies were administered weekly for three weeks. Animals were sacrificed on day 38. Tumor burden was assessed before each antibody dose and before sacrifice using bioluminescent imaging. (b) A single IV dose of CD3_F2BxBCMA was given to BALB/c mice or cynomolgus monkeys, and serum concentrations of the bispecific molecule were measured at multiple timepoints to estimate serum half-life.