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oai:dspace.mit.edu:1721.1/1515922023-08-01T03:06:45Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022

Wittrup, K. Dane Lax, Brianna Marie Massachusetts Institute of Technology. Department of Chemical Engineering 2023-07-31T19:51:04Z 2023-07-31T19:51:04Z 2023-06 2023-05-19T15:29:47.546Z https://hdl.handle.net/1721.1/151592 0000-0002-9727-7529 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 non-antagonistic 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 determination 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 both constructs was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments to elicit Fc effector-mediated Treg depletion. Through intratumoral administration of diphtheria toxin (DT) in Foxp3-DTR mice, we show that depletion of both intratumoral and intranodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-mediated restraint of priming in the tdLN. Lastly, we engineered a CTLA-4-targeted enzyme fusion as a potentially translatable approach for combined intratumoral and intranodal Treg depletion. Preliminary data suggest that CTLA-4 targeting increases local Treg death as a result of proximal enzymatic activity, but further characterization remains to be done. Overall, our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy - but that future therapies capable of depleting intranodal Tregs could show superior efficacy, even in the absence of CTLA-4 antagonism. Ph.D. Massachusetts Institute of Technology In Copyright - Educational Use Permitted Copyright retained by author(s) https://rightsstatements.org/page/InC-EDU/1.0/ Mechanistic Elucidation and Therapeutic Improvement of Anti-CTLA-4 Therapies Thesis application/pdf Doctoral Doctor of Philosophy Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="ded07ee7-b466-4bd2-9def-8257fa88875a"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Title>Mechanistic Elucidation and Therapeutic Improvement of Anti-CTLA-4 Therapies</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2023-06</PublicationDate> <Authors> <Author> <DisplayName>Lax, Brianna Marie</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>https://rightsstatements.org/page/InC-EDU/1.0/</License> <Abstract>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 non-antagonistic 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 determination 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 both constructs was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments to elicit Fc effector-mediated Treg depletion. Through intratumoral administration of diphtheria toxin (DT) in Foxp3-DTR mice, we show that depletion of both intratumoral and intranodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-mediated restraint of priming in the tdLN. Lastly, we engineered a CTLA-4-targeted enzyme fusion as a potentially translatable approach for combined intratumoral and intranodal Treg depletion. Preliminary data suggest that CTLA-4 targeting increases local Treg death as a result of proximal enzymatic activity, but further characterization remains to be done. Overall, our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy - but that future therapies capable of depleting intranodal Tregs could show superior efficacy, even in the absence of CTLA-4 antagonism.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>