Yeast Surface Display for Protein Engineering: Library Generation, Screening, and Affinity Maturation

Kang, Byong H.; Lax, Brianna M.; Wittrup, K. Dane

doi:10.1007/978-1-0716-2285-8_2

Cited by 4 publications

(2 citation statements)

References 93 publications

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“…The Sso7d scaffold was generated from a small DNA-binding protein and has previously been used to make murine therapeutic binders ( 73 ). These CTLA-4 binders were selected for as previously described using a soluble form of murine CTLA-4 fused to the CH2-CH3 domains of the mIgG2c Fc (CTLA-4-Fc) ( 3 , 74 ). Several rounds of flow-assisted cell sorting (FACS) were performed to enrich for binders with the highest affinity to CTLA-4-Fc.…”

Section: Resultsmentioning

confidence: 99%

“…Naive libraries were sorted using magnetic bead selections followed by FACS. Affinity maturation was performed by mutagenesis of identified binder sequences, library generation, and subsequent sorting using standard methods as described elsewhere ( 74 ). Mutagenesis of sequences was performed using error-prone PCR (epPCR), and libraries were generated using two-piece homologous recombination in yeast.…”

Section: Methodsmentioning

confidence: 99%

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Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies

Lax

Palmeri

Lutz

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy—but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%