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Redirecting T Cells to Tumor Targets

Streamlining T-cell engager development with functionally diverse CD3-binding antibodies

Immunotherapies are powerful cancer treatments that help the immune system identify and destroy cancer cells. CD3 T-cell engagers — a type of immunotherapy — are bispecific or multi-specific antibodies that redirect cancer-killing T cells to tumor targets. They bind both T cells and cancer cells simultaneously, bringing the two cells in close proximity, resulting in T cell activation and killing of the target tumor cell.

The Challenge

Developing effective T-cell engagers requires discovery of two parental antibodies — a CD3-binding arm that fine-tunes T cell activation and a tumor-binding arm with high specificity for cancer cells. Pairs of parental antibodies that function effectively in concert with each other are rare, necessitating diverse panels of antibodies that can be paired and tested at scale to find optimal clinical candidates.

The Goal

Use our integrated antibody discovery, characterization, and engineering technologies to break the barriers of conventional discovery and develop a large panel of fully human CD3-binding antibodies.

The Outcome

A panel of fully human CD3-binding antibodies that is:

Diverse

  • A range of CD3 affinities
  • A range of T cell activation potencies
  • Multiple epitope bins containing human and cynomolgus (cyno) cross-reactive binders

Developable

  • Favorable biophysical properties, including high thermal stability and low hydrophobicity, self-association, and polyspecificity

Validated

  • Proof-of-concept CD3 x EGFR T-cell engagers had a range of T cell activation and tumor cell killing potencies with low cytokine release

SEQUENCE DIVERSITY

We used proprietary immunization strategies with intact human and/or cyno CD3 complexes to induce robust immune response in humanized mice. We screened more than five million single antibody-producing cells using our single-cell screening technologies and identified more than 2,000 CD3-specific hits.

We used single-cell sequencing to identify more than 500 unique antibody sequences from ~160 different clonal families. More than 170 of these unique sequences were from single-cell hits with cross-reactivity to human and cyno homologs. 

Bioinformatic analyses revealed diverse antibody sequences with a range of V genes and CDR3 lengths, optimized by natural selection through somatic hypermutation.

Sequences from antibodies specific to human, cyno, and human and cyno CD3 were recovered following immunization and single-cell screening.
Sequences from antibodies specific to human, cyno, and human and cyno CD3 were recovered following immunization and single-cell screening.

FUNCTIONAL DIVERSITY

Our CD3-binding antibodies show binding and functional diversity. High-throughput antibody expression and characterization revealed a broad range of CD3 affinities and T cell activation potencies.

Surface Plasmon Resonance (SPR)-coupled analysis of antibody affinity for human CD3 εγ and εδ subunits.
Diverse CD3 affinities with KD values from ~1 nM – 1 µM. Surface Plasmon Resonance (SPR)-coupled analysis of antibody affinity for human CD3 εγ and εδ subunits.
SPR-coupled kinetics of antibody binding to the human CD3 εδ subunit
A range of on and off rates with diverse target engagement times. SPR-coupled kinetics of antibody binding to the human CD3 εδ subunit.
Activation of naïve primary human CD4+ and CD8+ T cells measured by CD25 and CD69 upregulation using flow cytometry.
Broad T cell activation potencies with EC50 values from ~6 – 200 µM. Activation of naïve primary human CD4+ and CD8+ T cells measured by CD25 and CD69 upregulation using flow cytometry. Values are the concentration of each antibody needed to induce 50% of the maximal activation.
We used high-throughput SPR epitope binning of CD3-binding antibodies, revealing multiple epitope bins. Antibodies with cross-reactivity to human and cyno CD3 were found in multiple epitope bins, including those that were distinct from characterized, commercially available CD3-specific antibodies that are commonly used for T-cell engager development, such as UCHT1 and SP34-2.
High-throughput SPR-based epitope binning of CD3-binding antibodies reveals multiple epitope bins.
High-throughput SPR-based epitope binning of CD3-binding antibodies reveals multiple epitope bins.

DEVELOPABILITY

High-throughput antibody assessment identified antibodies with promising developability traits, including:

Surface hydrophobicity, self-association, and polyspecificity analysis of CD3-specific antibodies.
Biophysical analysis of CD3-specific antibodies. (A) Relative surface hydrophobicity measured by analytical hydrophobic interaction chromatography (aHIC), (B) Self-association assessed by affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS), (C) Polyspecificity assessed by baculovirus particle enzyme-linked immunosorbent assay (BVP-ELISA).

VALIDATION

Engineering antibodies that potently eliminate cancer cells without inducing toxicity has been a significant challenge limiting T-cell engager development. In proof-of-concept experiments, we used our OrthoMabTM bispecifics platform to generate CD3 x EGFR T-cell engagers. High-throughput functional and biophysical assays identified developable and potent T-cell engager molecules that maintained low levels of cytokine production.

Broad T cell activation potencies measured for bispecific antibodies.
Broad T cell activation potencies with EC50 values from ~2 pM – 2 nM. Effector T cells were incubated with target CHO-K1 cells expressing low or medium levels of EGFR for six hours at a ratio of 7:1. The concentration of each bispecific antibody needed to induce 50% of the maximal activation of NFAT reporter Jurkat T cells is reported.
Broad tumor cell killing potencies measured for bispecific antibodies.
Broad tumor cell killing potencies with EC50 values from ~0.01 – 1 nM. Unactivated human T cells were incubated with target cells at a ratio of 10:1. Cell killing was measured at 48 hours using an xCELLigence assay. The concentration of each bispecific antibody needed to induce 50% of the maximal T cell-mediated killing of HeLa cells is reported.
A range of cytokine release measured for bispecific antibodies.
A range of cytokine release. Unactivated human T cells were incubated with target cells at a ratio of 10:1. Cytokines were measured after 48 hours of incubation with varying levels of bispecific antibodies. Cytokine concentrations were calculated for each antibody at 60% and 80% HeLa cell killing. The concentration of bispecific antibody needed to reach 60% tumor cell killing was 0.02 – 5 nM and was 0.07 -5 nM to reach 80% tumor cell killing.

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