Supplementary Figure S3. Raw pixel intensities for western blots in Figure 2

Supplementary Table S1. List of antibodies

Abstract Brentuximab vedotin, a CD30-directed antibody–drug conjugate (ADC), is approved for clinical use in multiple CD30-expressing lymphomas. The cytotoxic payload component of brentuximab vedotin is monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. Preclinical results provided here demonstrate that treatment of cancer cells with brentuximab vedotin or free MMAE leads to a catastrophic disruption of the microtubule network eliciting a robust endoplasmic reticulum (ER) stress response that culminates in the induction of the classic hallmarks of immunogenic cell death (ICD). In accordance with the induction of ICD, brentuximab vedotin–killed lymphoma cells drove innate immune cell activation in vitro and in vivo. In the “gold-standard” test of ICD, vaccination of mice with brentuximab vedotin or free MMAE-killed tumor cells protected animals from tumor rechallenge; in addition, T cells transferred from previously vaccinated animals slowed tumor growth in immunodeficient mice. Immunity acquired from killed tumor cell vaccination was further amplified by the addition of PD-1 blockade. In a humanized model of CD30+ B-cell tumors, treatment with brentuximab vedotin drove the expansion and recruitment of autologous Epstein-Barr virus–reactive CD8+ T cells potentiating the activity of anti–PD-1 therapy. Together, these data support the ability of brentuximab vedotin and MMAE to drive ICD in tumor cells resulting in the activation of antigen-presenting cells and augmented T-cell immunity. These data provide a strong rationale for the clinical combination of brentuximab vedotin and other MMAE-based ADCs with checkpoint inhibitors.

Supplementary figures and table

Abstract Antibody-drug conjugates (ADCs) combine the targeting specificity of antibodies with powerful cytotoxic payloads to direct antigen-specific tumor cell killing. Multiple ADCs have shown single-agent clinical activity against solid and hematological cancers and are currently being evaluated in combination with immune checkpoint inhibitors (CPIs). Therapeutic benefit from CPIs relies on the presence and quality of responding antitumor T cells, both in the tumor and from new T cell priming and expansion outside the tumor. Select cytotoxic ADC payloads, like monomethyl auristatin E (MMAE), have the potential to amplify T cell responses through immunogenic cell death (ICD) and, as such, may be optimal combination partners with CPIs. However, ADC payloads released in the tumor (bystander drug) or released systemically also have the potential to negatively impact T cell viability, proliferation, and priming. The compatibility of different ADC payload chemotypes (Auristatins, camptothecins (CPTs), Pyrrolobenzodiazepine (PBDs), Tubulysins) with CPIs may depend on both the direct and indirect effects of payloads and their associated free-drugs on immune cells. Here, we determined the relative potencies of ADC payload free-drugs MMAE and DXd on activated human and murine T cells in vitro and in a model of xeno-graft versus host disease (xeno-GVHD) to evaluate the impact of ADC bystander effects on human immune cell expansion and function in vivo. In vitro, ADC payloads showed a range of potencies on activated T cells, with IC50 values skewing lower for CD4 compared to CD8 T cells. For each payload, murine T cells were significantly less sensitive than human T cells, which may have implications for modeling combinations of ADCs and CPIs. Mouse CD8 T cells were ~10-fold less sensitive to MMAE and 20-25-fold less sensitive to clinical CPT payloads compared to human T cells. In an in vivo model of human immune cell activation, xeno-GVHD, bystander effects from a non-targeted MMAE-ADC did not impair the expansion of human immune cells, however, a non-targeted DXd-ADC reduced CD4 T cells and robustly reduced populations of non-T cells in spleens. Consistent with reduced immune cell expansion, the DXd-ADC significantly slowed the xeno-GVHD disease course compared to the MMAE-ADC treated and control groups. These data suggest that the most commonly used ADC payload classes, MMAE and DXd, may have different direct effects on proliferating human immune cells as bystander free-drugs, which may have clinical relevance for combinations of ADCs with CPIs. Continued research exploring the balance between ADC potency on tumor cells, direct and indirect effects of payloads on immune cells, and dose timing and sequencing could help maximize the potential of ADCs in combination with immunotherapies. Citation Format: Reice D. James, Michelle L. Ulrich, J Hartsuyker, Alyson J. Smith, Ryan A. Heiser. Evaluating the potency of antibody-drug conjugate (ADC) free-drugs on T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2604.

Supplementary Figure S1. Overview of ICD

Supplementary Figure S4. pIRE, pJNK, and CHOP upregulation in lymphoma lines treated with brentuximab vedotin or MMAE

Supplementary Figure S2. ICD hallmarks observed from tumor cell lines treated with brentuximab vedotin or MMAE

Background The presence of regulatory T cells (Tregs) in solid tumors attenuates the ability of immune cells to mediate an effective response to cancer. Expansion and activation of intratumoral Tregs is a proposed mechanism of PD-1/PD-L1 checkpoint inhibition resistance and elimination of these cells could facilitate an immune response to tumors. Single cell transcriptomic and flow cytometric analysis of tumor-associated immune cells revealed enhanced expression of CD30 on an activated subset of Tregs, suggesting that treatment with an anti-CD30 molecule could selectively deplete this subset of intratumoral Tregs. Moreover, anti-PD1 treatment of intratumoral T cells selectively enhanced expression of CD30 on Tregs compared with either CD4+ or CD8+ T cells, suggesting compatibility with immune checkpoint inhibitor therapy. The anti-CD30 antibody-drug conjugate (ADC) brentuximab vedotin is approved for the treatment of advanced classical Hodgkin lymphoma and other CD30-expressing lymphomas. It is currently being tested in combination with pembrolizumab for the ability to deplete Tregs and increase immune checkpoint activity in anti-PD-1-refractory metastatic melanoma and NSCLC (NCT04609566). Methods Preliminary analysis of IHC and RNA-seq data from paired biopsies in responder patients suggests increased tumor-infiltrating CD8+ T cells post treatment. SGN-35T, an anti-CD30 ADC with the same antibody (cAC10) and cytotoxic payload (monomethyl auristatin E) as brentuximab vedotin, contains a novel tripeptide linker comprised of D-leucine-alanine-glutamate (DLAE) and was developed to improve the tolerability profile while leveraging the known activity of brentuximab vedotin. Results In addition to cytotoxic activity on CD30-expressing tumor cells, SGN-35T depleted CD30-expressing Tregs in vitro, while in contrast CD8+ T cells were not affected. This may be due to the variable expression of drug efflux transporters which differs across T cell subsets and activation states, with Tregs demonstrating limited efflux capacity in contrast to naive CD8+ T cells and memory CD8+ T cells. Conclusions Thus, the cytotoxic activity of SGN-35T on CD30-expressing Tregs is likely a combination of the enrichment of CD30 expression on activated Tregs coupled with reduced drug efflux capacity. Together, these data support future clinical investigation of SGN-35T in combination with anti-PD-1/PD-L1 checkpoint inhibitors in solid tumors.

TIGIT is an immune checkpoint receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells, and natural killer (NK) cells. TIGIT has emerged as an attractive target for antitumor therapies, due to its proposed immunosuppressive effects on lymphocyte function and T cell activation. We generated an anti-TIGIT monoclonal antibody (mAb) that binds with high affinity to human, non-human primate, and murine TIGIT and through multiple experimental methodologies demonstrated that checkpoint blockade alone is insufficient for antitumor activity. Generating anti-TIGIT mAbs with various Fc backbones we show that muting the Fc-Fcγ receptor (FcγR) interaction failed to drive antitumor activity, while mAbs with Fc functional backbones demonstrate substantial antitumor activity, mediated through activation of antigen-presenting cells (APCs), T cell priming, and NK-mediated depletion of suppressive Tregs and exhausted T cells. Further, nonfucosylation of the Fc backbone resulted in enhanced immune responses and antitumor activity relative to the intact IgG1 backbone. The improved activity correlated with the biased FcγR interaction profile of the nonfucosylated anti-TIGIT mAb, which supports that FcγRIIIa binding with decreased FcγRIIb binding favorably activates APCs and enhances tumor-specific CD8+ T cell responses. The anti-TIGIT mAbs with intact FcγR interacting backbones also demonstrated synergistic enhancement of other standard antitumor treatments, including anti-PD-1 treatment and a model monomethyl auristatin E antibody–drug conjugate. These findings highlight the importance of the anti-TIGIT mAb’s Fc backbone to its antitumor activity and the extent to which this activity can be enhanced through nonfucosylation of the backbone.

Agonist antibodies targeting 4-1BB (CD137) effectively costimulate cytotoxic T cells and are active in preclinical models of cancer. However, the clinical development of these agents has been hampered by limited efficacy and/or poor tolerability at active doses. To overcome the efficacy and safety limitations of this approach, SGN-BB228, a first-in-class, investigational CD228 × 4-1BB costimulatory Antibody Anticalin® bispecific (MabcalinTM) was created. SGN-BB228 is designed to target CD228 (melanotransferrin), a GPI-anchored oncofetal membrane protein with limited normal tissue expression, but high prevalence and expression in melanoma, mesothelioma, lung cancer, and other tumor types. SGN-BB228 is designed to provide a potent costimulatory bridge between tumor-specific T cells and tumor cells, improving and limiting T cell-mediated cytotoxicity to tumors, potentially expanding the therapeutic window for 4-1BB agonism. Here we describe the anti-tumor activity and pharmacodynamic effects of SGN-BB228 in vivo using humanized mouse tumor models, and the in vitro costimulatory effect of SGN-BB228 in a tumor cell line-based model of functional T cell exhaustion. In vivo, SGN-BB228 improved the quality and magnitude of the cytotoxic T cell response within CD228-expressing tumors, augmenting anti-tumor immunity. In vitro, 4-1BB costimulation provided by SGN-BB228 elicited proliferation, and cytokine production from functionally exhausted human T cells when in culture with CD228-expressing tumor cell lines engineered to engage the T cell receptor. Functionally exhausted T cells in this system are a mixed population that share phenotypic markers and single cell transcriptional signatures associated with either progenitor exhausted T cells (TPEX) or terminally exhausted T cells (TEX). Interestingly, in this system, anti-PD-1 (nivolumab) failed to reinvigorate functionally exhausted T cells despite high PD-L1 expression by tumor cells. The unique ability of 4-1BB costimulation provided by SGN-BB228 to improve T cell activity in this model suggests the potential to drive immunomodulation in circumstances where PD-1 blockade fails. Together these data highlight SGN-BB228, a first-in-class, investigational CD228 × 4-1BB costimulatory Antibody Anticalin® bispecific with potent and CD228-conditional 4-1BB costimulatory activity with therapeutic potential in multiple solid tumor types. These data support the first-in-human phase 1 clinical trial of SGN-BB228 in advanced melanoma and other solid tumors (NCT05571839), which is currently recruiting. Citation Format: Barrett Updegraff, Johannes Urban, James Mutschler, Gregory L. Szeto, Brian P. O'Connor, Shyra J. Gardai, Ryan A. Heiser. SGN-BB228, a CD228-directed costimulatory antibody anticalin® bispecific provides potent and conditional 4-1BB costimulation to T cells in vivo and in an in vitro model of T cell exhaustion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5676.

Background Antibody drug conjugates (ADCs), that pair highly cytotoxic drugs with a tumor targeting antibody, have become a mainstay of many cancer treatment paradigms. Currently approved ADCs utilize payloads that disrupt tubulin (including monomethyl auristatins (MMAE and MMAF), inhibit topoisomerase 1 (DXd and SN-38) or induce DNA cleavage (ozogamicin). Primarily these drugs have been studied for their direct cytotoxic effects on tumor cells, but many are highly permeable and have the potential to affect other intra-tumoral cells, including immune cells. These effects could adversely impact anti-tumor immunity and/or synergy with immunotherapies, but to date has been under studied. Methods To determine how these various free drugs effect immune cells within a tumor microenvironment we compared the sensitivity of various innate immune cells (peripheral NK cells, IL2/IL15 expanded and activated NK cells, in vitro derived macrophages in various polarization states (M0, M1, TAM) and myeloid derived suppressor cells (MDSC)) to killing directed by tubulin disruptor payloads (MMAE, MMAF) various camptothecin (CPTs) topoisomerase 1 inhibiting payloads or a highly potent DNA crosslinking payload pyrrolobenzodiazepine (PBD). We assessed how payload treatment effects hallmark innate immune activities of these cells. For macrophages, we queried how payloads changed the ability to respond to LPS challenge and for MDSC we assessed ability to suppress T cell proliferation after CD3/28 stimulation. Induction of cytokine production directly from tumor cells in response to payloads was also assessed. Results The PBD compound proved to be highly cytotoxic to all cell types, while MMAF generally did not demonstrate cytotoxicity to any of the cells tested, likely due to the low permeability of the drug. Innate immune cells demonstrated differential sensitivity to killing by the CPTs vs. MMAE. Activated NK cells showed the highest sensitivity, followed by peripheral NK cells and then MDSCs, with the least sensitive being macrophages. Generally, all cell types were more sensitive to killing by CPTs than MMAE. When evaluating activity, pre-treatment with MMAE unexpectedly drove increased macrophage activation while CPT treatment resulted in decreased activation. For MDSCs, it was noted that pre-treatment with certain payloads caused less suppression of activated T cells. And finally, robust cytokine induction was observed from several tumor cell lines that was quite varied based on payload used and cell line. Conclusions These data begin to elucidate the effects of various ADC payloads on intratumoral bystander innate immune cells and expand our understanding of the wider effects of ADCs on the tumor microenvironment. Acknowledgements We would like to acknowledge Ryan Lyski, Joe Hamilton, Philip Moquist and Svetlana Doronina for their provision of study materials and chemical insight.

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis by preventing excessive inflammation in normal tissues. In cancer, Tregs hamper antitumor immunosurveillance and may be a resistance mechanism to anti-PD-1 therapies. In preclinical cancer models, Treg depletion enhances antitumor cytotoxic immune responses, but systemic and persistent Treg removal can elicit immunopathology. Therapeutic strategies that selectively deplete highly suppressive activated intratumoral Tregs may avoid immune-related toxicities and amplify the activity of antitumor CD8 T cells during PD-1 blockade. CD30 is a member of the TNF receptor superfamily and is expressed by a subset of activated lymphocytes and various lymphomas. Here, transcriptomics and flow cytometry data from tumor and peripherally derived Tregs demonstrated CD30 is enriched on intratumoral Tregs. Among peripheral Tregs, CD30 expression was associated with an activated effector phenotype (FoxP3hiCD45RA-, fraction II). Further, treatment of dissociated NSCLC tumor spheroids with anti-PD-1 resulted in upregulated CD30 expression by Tregs but not intratumoral CD4 or CD8 T cells, which is consistent with Treg reactivation and supports CD30 as an activated Treg target. In vitro, CD30 expression on Tregs was dependent on signaling through the TCR and was significantly amplified by IL-2 and IL-15, suggesting CD30 may identify a subpopulation of activated antigen-specific Tregs. Brentuximab vedotin (BV) is an antibody-drug conjugate approved for clinical use in multiple types of lymphomas including cHL and PTCL. BV consists of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE). The activity of BV in lymphomas is thought to primarily result from tumor-directed intracellular MMAE release leading to apoptosis, though multiple immunomodulatory effects of BV have also been suggested. We have demonstrated through in vitro and in vivo preclinical model systems of human T cell activation that BV can selectively deplete CD30-expressing Tregs, amplifying cytotoxic CD8 T cell expansion. These and Treg CD30 expression data motivated the clinical exploration of BV plus pembrolizumab in metastatic PD-1 refractory melanoma and NSCLC (NCT04609566). Consistent with preclinical data, preliminary biomarker analysis from these studies showed a small but significant decrease in Tregs (CD4+CD25+CD127low/-) in peripheral blood after treatment with BV plus pembrolizumab. Further, limited on-treatment biopsies showed a trend toward increased intratumoral CD8:Treg (FoxP3) ratio in some patients receiving combination therapy. These data support the ongoing evaluation of BV in combination with PD-1 inhibitors in solid tumors and a potential additional mechanism of action for BV. Citation Format: Ryan A. Heiser, Bryan M. Grogan, Reice D. James, Michelle L. Ulrich, Jason D. Berndt, Brian P. O'Connor, Shyra J. Gardai, Hailing Lu, Scott M. Knowles. CD30 is a marker of activated effector regulatory T cells in solid tumors providing clinical rationale for the combination of brentuximab vedotin and PD-1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3253.

Background Agonist antibodies targeting 4-1BB (CD137) effectively costimulate cytotoxic T cells and are active in preclinical models of cancer. However, clinical development of these agents has been hampered by limited efficacy and/or poor tolerability at active doses.1,2 To overcome the efficacy and safety limitations of this approach, SGN-BB228, a first-in-class, investigational CD228/4-1BB Antibody Anticalin® bispecific was created. SGN-BB228 targets CD228 (melanotransferrin, p97), a glycosylphosphatidylinositol-anchored membrane protein with limited normal tissue expression, but high and prevalent expression in melanoma, mesothelioma, lung cancer and other tumor types.3,4 SGN-BB228 is designed to provide a potent costimulatory bridge between tumor-specific T cells and tumor cells, improving and constraining T cell mediated cytotoxicity to tumors, potentially expanding the therapeutic window for 4-1BB agonism. Methods Here we describe the expression profile of CD228 in cancer and normal tissues and preclinical activity of SGN-BB228 across reporter cell and primary T cell-based assays. Results SGN-BB228 is comprised of a hinge-stabilized (S228P), Fc-null (FALA) fully human IgG4 antibody specific for CD228 connected to 4-1BB-targeting Anticalin® proteins5 via C-terminal heavy-chain fusions. The proposed mechanism of action (MOA) for SGN-BB228 is CD228-conditional clustering of 4-1BB on antigen experienced tumor-specific T cells, resulting in enhanced activation and cellular cytotoxicity. Expression analysis across cancer and normal tissues demonstrates CD228 is a tumor associated antigen prevalent in melanoma, mesothelioma, lung cancer and other tumor types with minimal normal tissue expression. Preclinical testing of SGN-BB228 in vitro shows potent CD228-conditional 4-1BB stimulation and cytotoxic T cell activation across a range of assay systems. In the presence of CD228-expressing tumor cells, but not CD228-negative tumor cells, SGN-BB228 drove dose-dependent amplification of NFkB signaling using a 4-1BB reporter cell system. SGN-BB228 also consistently drove potent CD228-conditional costimulatory activity in assays using primary T cells or whole peripheral blood mononuclear cells (PBMCs) receiving different forms of T cell receptor stimulation. The CD228-conditional activity of SGN-BB228 consistently outperformed a clinical benchmark antibody, even in the presence of antibody-clustering FcgRs expressed by PBMC. Conclusions Together these data introduce SGN-BB228, a first-in-class, investigational CD228/4-1BB costimulatory Antibody Anticalin® bispecific with potent and CD228-conditional 4-1BB costimulatory activity with therapeutic potential in multiple solid tumor types. These data support future clinical study of SGN-BB228 in a first-in-human Phase 1 trial. References Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res. 2017;23(8):1929–36. Segal NH, He AR, Doi T, et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018;24(8):1816–23. Brown JP, Nishiyama K, Hellström I, Hellström KE. Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies. J Immunol 1981;127(2):539–546. Smith LM, Nesterova A, Alley SC, Torgov MY, Carter PJ. Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97. Mol Cancer Ther 2006;5(6):1474–1482. Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res. 2019;25(19):5878–89.

Background Enfortumab vedotin (EV) is a first-in-class Nectin-4-directed antibody-drug conjugate (ADC) with demonstrated improved overall survival in patients with previously treated advanced-stage urothelial carcinoma.1 EV is comprised of a fully human Nectin-4-directed monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) by a protease cleavable maleimidocaproyl-valine-citrulline linker. EV has a multifaceted mechanism of action. Previously, we demonstrated that EV induces antitumor activity in vitro via direct cytotoxicity on Nectin-4-expressing malignant cells and indirect bystander activity on neighboring Nectin-4­ negative cells, both of which are mediated by MMAE release within target cells. Here, we expand upon the mechanism of action and show EV induces tumor cell killing in a manner leading to immunogenic cell death (ICD) and improves antitumor responses when combined with checkpoint inhibitors. Methods The ability of EV to induce hallmarks of ICD was evaluated in vitro in Nectin-4-expressing human urothelial carcinoma cell lines. Immune activation associated with ICD was assessed in vitro in monocytes co-cultured with EV-treated tumor cells and in vivo by immunohistochemistry, RNA-seq, flow cytometry, and immune cytokine profiling. The effects of EV plus anti-PD-1 on tumor growth inhibition, the tumor microenvironment, and immune memory were evaluated in syngeneic mouse models engineered to express human Nectin-4. Antitumor immune memory was also assessed in mice vaccinated with EV-treated cells. Results In vitro, EV induced ICD via MMAE-mediated microtubule disruption and concomitant endoplasmic reticulum (ER) stress, as evidenced by increased phosphorylation of JNK, extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. Xenograft tumors treated with EV demonstrated upregulation of MHC genes as well as genes involved in ER stress, autophagy, and type I interferon response. Additionally, there were noted increases in both macrophages and dendritic cells along with cytokines involved in chemoattraction and T-cell stimulation. Consistent with ICD induction, vaccination with EV-treated Nectin-4-expressing tumor cells promoted antitumor immunity and provided protection against tumor rechallenge. Lastly, the combination of EV with PD-1 inhibition improved antitumor activity and durable immunity in vivo, consistent with complementary modes of action of these two anticancer agents. Conclusions These data provide insight into the clinical activity observed with EV and bolster the scientific rationale to combine EV with checkpoint inhibitors, which is currently an area of active clinical investigation across multiple studies.2-6 References Powles T, Rosenberg JE, Sonpavde GP, Loriot Y, Duran I, Lee JL, et al. Enfortumab Vedotin in Previously Treated Advanced Urothelial Carcinoma. N Engl J Med. 2021;384(12):1125–35. Epub 2021/02/13. doi:10.1056/NEJMoa2035807. PubMed PMID: 33577729; PubMed Central PMCID: PMCPMC8450892. Friedlander TW, Milowsky MI, Bilen MA, Srinivas S, McKay RR, Flaig TW, et al. Study EV-103: Update on durability results and long term outcome of enfortumab vedotin + pembrolizumab in first line locally advanced or metastatic urothelial carcinoma (la/mUC). Journal of Clinical Oncology 2021;39(15_suppl):4528. doi: 10.1200/JCO.2021.39.15_suppl.4528. Galsky MD, Necchi A, Shore ND, Plimack ER, Jia C, Sbar E, et al. KEYNOTE-905/EV-303: Perioperative pembrolizumab or pembrolizumab plus enfortumab vedotin (EV) and cystectomy compared to cystectomy alone in cisplatin-ineligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol. 2021;39(6_suppl):TPS507. doi: 10.1200/JCO.2021.39.6_suppl.TPS507. Heijden MSVD, Gupta S, Galsky MD, Derleth CL, Lee S, Kataria RS, et al. Study EV-302: A two-arm, open-label, randomized controlled phase 3 study of enfortumab vedotin in combination with pembrolizumab versus chemotherapy in previously untreated advanced urothelial carcinoma (aUC) (trial in progress). J Clin Oncol 2022;40(6_suppl):TPS589. doi: 10.1200/JCO.2022.40.6_suppl.TPS589. Hoimes CJ, Bedke J, Loriot Y, Nishiyama H, Fang X, Kataria RS, et al. KEYNOTE-B15/EV-304: Randomized phase 3 study of perioperative enfortumab vedotin plus pembrolizumab versus chemotherapy in cisplatin-eligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol 2021;39(15_suppl):TPS4587. doi: 10.1200/JCO.2021.39.15_suppl.TPS4587. ClinicalTrials.gov [Internet] Bethesda (MD): U.S. National Library of Medicine. 2000 – . ClinicalTrials.gov Identifier: NCT04960709. Treatment Combination of Durvalumab, Tremelimumab and Enfortumab Vedotin or Durvalumab and Enfortumab Vedotin in Patients With Muscle Invasive Bladder Cancer Ineligible to Cisplatin or Who Refuse Cisplatin (VOLGA). 2021 Jul 14 [cited 2022 Jul 22]. Available from: https://clinicaltrials.gov/ct2/show/NCT04960709. Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen, Astellas, or the external testing facilities that conducted the studies.

Background Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs. Methods Here we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo. Results Treatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression. Conclusions Together, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs. Acknowledgements The authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation. Ethics Approval Animals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

Abstract Brentuximab vedotin (BV) is an antibody-drug conjugate (ADC) directed against CD30, a TNF receptor superfamily (TNFRSF) member highly expressed on Reed Sternberg cells in Hodgkin lymphoma (HL) and also commonly expressed in a number of other lymphoid malignancies such as ALCL and CTCL. BV consists of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. MMAE-based ADC antitumor activity primarily results from intracellular payload release, leading to mitotic arrest and apoptotic cell death, although secondary MOAs may exist. CD30 is mostly absent on resting peripheral lymphocytes, but is known to be transiently upregulated on both CD4+ and CD8+ T cells following activation. Recently, CD30 was identified, by separate research groups, as a marker differentially upregulated by human intratumoral T regulatory cells (Tregs). This recent observation raises the possibility that BV could target and eliminate intratumoral CD30+ Tregs. Abundant evidence shows that tumor-specific CD8+ T cells are paramount to antitumor immunity. In contrast, tumor-resident T regulatory cells have been shown to counteract immunosurveillance and promote tumor escape. Given that CD30 may be expressed on both activated CD8+ T cells and intratumoral Tregs, we explored the outcome of BV treatment on each cell type. In this work, we show that BV directly depleted inducible and primary CD30+ Tregs in vitro, in a dose-dependent manner, while CD30+ CD8+ T cells were unaffected. Moreover, BV selectively depleted Tregs in a Treg:CD8 T cell co-culture suppression assay, resulting in expansion of proliferating CD8+ T cells. In vivo, using a humanized mouse model of graft-versus-host disease (xeno-GVHD), treatment of mice with BV significantly reduced splenic Treg numbers, while amplifying total xeno-reactive CD8+ cytotoxic T cells. In an effort to understand BV's selective impairment of Tregs, we evaluated CD30 expression following in vitro activation with CD3/CD28. Freshly isolated Tregs showed notably accelerated CD30 expression kinetics along with significantly higher peak receptor number compared to CD8+ T cells. Furthermore, assays confirmed that the heightened receptor expression on Tregs translated into increased BV internalization and drug-linker cleavage. Finally, CD8+ T cells were much more efficient at effluxing rhodamine than Tregs, suggesting lowered drug accumulation and exposure over time. Together, these data raise the novel possibility that brentuximab vedotin may be able to positively impact the Treg:CD8 T cell balance in the tumor microenvironment through selective depletion of CD30+ Tregs, but not activated CD8+ T cells. Citation Format: Ryan A. Heiser, Bryan M. Grogan, Luke S. Manlove, Shyra J. Gardai. CD30+T regulatory cells, but not CD30+CD8 T cells, are impaired following brentuximab vedotin treatment in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1789.