{
  "abstract": "Background Currently approved monospecific antibody checkpoint inhibitors are often insufficiently effective for all patients and/or indications. Thus, investigators are interested in targeting multiple signaling pathways and/or cell types to enhance clinical outcomes, often in the form of multispecific antibody treatments (‘multispecifics’). Many of these multispecifics target the T cell as T cell engagers (TCEs), with the majority targeting CD3 and a tumor-associated antigen to activate T cell-dependent cellular cytotoxicity. Co-stimulation by targeting CD28 or CD137 in addition to CD3 has recently shown promise. However, the large number of possible topologies and added complexity for manufacturing necessitate the development and application of a robust set of complementary technologies that facilitate generation and allow for functional screening of large multispecific panels.Methods Co-stimulatory antibodies against CD28 were identified from synthetic IgG and heavy chain-only antibody (HCAb) libraries using a yeast-based antibody platform. Discovered CD28 antibodies were subsequently optimized to generate a panel of leads with varied epitope breadth, affinity, and good developability. Bispecific CD28 x HER2 leads were subsequently generated using a number of internally developed chain pairing technologies. 1 3 Bispecific CD28 antibodies were then assessed for their ability to stimulate T cell-dependent cellular cytotoxicity (TDCC) when combined with a broad affinity panel of CD3 antibodies.2 Results Mutation sets have been identified that drive greater than 95% desired HC-LC pairing and HC-HC heterodimerization when producing bispecific antibodies in a single transfection system. An alternative set of mutations is amenable to producing large arrays of bispecific antibodies using a chain exchange reaction. A format-diverse panel of anti-CD28 antibodies has been discovered and used to generate bispecific antibodies using our pairing mutation sets. The resulting bispecific antibodies have been shown to provide costimulatory signal and enhance tumor cell killing in TDCC assays when paired with an anti-CD3 bispecific antibody.Conclusions We have developed an exemplary set of technologies that demonstrate the ability to direct desired antibody chain pairing (HC-HC and HC-LC), isolate and engineer single-domain antibodies, as well as generate large panels of multispecific antibodies with diverse topologies from a limited number of input molecules. Specifically, we illustrate these capabilities in the context of TCE multispecifics that leverage our affinity- and developability-optimized anti-CD3 and anti-CD28 antibody panels.References Barlow KA, Battles MB, Brown ME, Canfield K, Lu X, Lynaugh H, Sivasubramanian A. Design of orthogonal constant domain interfaces to aid proper heavy/light chain pairing of bispecific antibodies. MABs. 2025;17.Liu, CY, Ahonen CL, Brown, ME, Zhou L, Welin M, Krauland EM, Pejchal R, Widboom, P, Battles MB. Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity. MABs. 2023;15.Pejchal R, Cooper, B, Brown ME, Vásquez M, Krauland EM. Profiling the biophysical developability properties of common IgG1 Fc effector silencing variants. Antibodies. 2023;12.",
  "authors": [
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Nathan Sharkey"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Cory Ahonen"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Melissa Durkin"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Andrew Avery"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Michael Battles"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Beth H Sharkey"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Kyle Barlow"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Arvind Sivasubramanian"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Caitlin Stein"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Bradley Lunde"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Kevin Schutz"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Robert Pejchal"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Bianka Prinz"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Paul Widboom"
    },
    {
      "affiliations": [
        "Adimab, Lebanon, NH, USA"
      ],
      "name": "Eric Krauland"
    }
  ],
  "title": "1024 Rapid and efficient generation of format-diverse co-stimulatory CD28 and CD3 multispecific antibody panels for T cell engagement via complementary technologies",
  "uid": "f9cd1405-e16a-5bb2-9f36-f3d02a7842f0"
}