Article Text
PDF
Abstract
Background SGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex.
Methods B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model.
Results Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent.
Conclusion The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.
- Drug Evaluation, Preclinical
- Drug Therapy, Combination
- Therapies, Investigational
- Translational Medical Research
- Tumor Microenvironment
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
- Drug Evaluation, Preclinical
- Drug Therapy, Combination
- Therapies, Investigational
- Translational Medical Research
- Tumor Microenvironment
WHAT IS ALREADY KNOWN ON THIS TOPIC
B7-H4 is an immune checkpoint ligand known to have elevated expression on a variety of solid tumors, but the mechanism of action and antitumor activity of the B7-H4-directed antibody–drug conjugate SGN-B7H4V had yet to be characterized.
WHAT THIS STUDY ADDS
Our results suggest that SGN-B7H4V internalizes following binding to B7-H4, and kills tumor cells via monomethyl auristatin E -mediated direct cytotoxicity as well as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. SGN-B7H4V demonstrated robust antitumor activity in in vivo xenograft models of breast and ovarian cancer. SGN-B7H4V in combination with an anti-programmed cell death-1 (PD-1) agent led to improved antitumor activity and elicited durable immune memory.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study demonstrates that SGN-B7H4V has promising antitumor activity in preclinical models as a monotherapy and in combination with an anti-PD-1 agent in vivo, and is currently being evaluated in clinical trials as a treatment for certain solid tumors.
Background
SGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising three components: a fully human IgG1 monoclonal antibody (mAb) that binds to the immune checkpoint ligand B7-H4 (VTCN1) on the cell surface, approximately four molecules (ie, drug–antibody ratio (DAR) of ~4) of the microtubule-disrupting agent monomethyl auristatin E (MMAE), and a protease-cleavable maleimidocaproyl valine-citrulline (mc-vc) linker.1 This vedotin linker-payload system has been clinically validated in several approved therapeutics including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin.2–4 SGN-B7H4V is the first vedotin ADC targeting an immune checkpoint ligand and is currently under investigation in a phase 1, first-in-human trial (SGNB7H4V-001, NCT05194072) evaluating SGN-B7H4V monotherapy in patients with select advanced tumors.5
B7-H4 is a member of the B7 family of immune checkpoint ligands and binds to an unknown receptor on T cells, and like the widely known programmed death-ligand 1 (PD-L1)/B7-H1 checkpoint ligand, has been shown to negatively regulate T-cell function by inhibiting T-cell proliferation and cytokine production.6–9 In contrast to its limited expression in normal tissues, B7-H4 is elevated across a broad range of solid tumors, including ovarian cancer, breast cancer, endometrial carcinoma, cholangiocarcinoma, gallbladder carcinoma, and squamous non-small cell lung cancer (NSCLC) where it is hypothesized to help tumor cells avoid immune surveillance.9–16 Consistent with its immunosuppressive function, B7-H4 expression in tumors has been associated with lower survival rates, advanced clinical stage, increased lymph node involvement, and decreased tumor T-cell infiltration.17 Accordingly, targeting B7-H4-expressing tumor cells and removal of the B7-H4/ADC complex from the cell surface via internalization may relieve B7-H4-mediated immunosuppression.6–8 18
Like other vedotin ADCs, SGN-B7H4V is hypothesized to have a multimodal mechanism of action (MOA) and may kill target cells through both MMAE-mediated cytotoxicity and antibody-mediated effector functions. Vedotin ADCs can induce MMAE-mediated direct cytotoxicity by preferentially localizing to target cells and releasing MMAE following internalization.19–22 Once MMAE is released into the cytoplasm, it binds and disrupts the microtubule network, leading to cell cycle arrest and apoptosis.22–24
MMAE released from vedotin ADCs has also been shown to kill cells in a manner consistent with immunogenic cell death (ICD), which may promote activation and recruitment of immune cells to tumors.25–27 The ability of vedotin ADCs to induce ICD bolsters the rationale for combining them with immunotherapies, such as anti-programmed cell death-1 (PD-1) agents, to augment antitumor immune responses. This is supported by clinically meaningful responses observed when vedotin ADCs are combined with PD-1 inhibitors.28
Here, we characterize B7-H4 expression by various tumor types, investigate the MOA of SGN-B7H4V in several preclinical models, and evaluate the antitumor activity of SGN-B7H4V alone and in combination with an anti-PD-1 agent. Our results support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.
Methods
Immunohistochemistry staining for B7-H4 on human tumor samples and validation of anti-B7-H4 mAb clone D1M8I
Immunohistochemistry (IHC) staining for B7-H4 was performed with 5 µg/mL rabbit antibody clone D1M8I (Cell Signaling #14572, Danvers, Massachusetts, USA) on formalin-fixed, paraffin-embedded (FFPE) human cancer tissue microarrays (TMAs) (US Biomax, Derwood, Maryland, USA) and full tissue sections. Isotype-matched rabbit IgG (Abcam, clone EPR25a #ab172730, Waltham, Massachusetts, USA) was used as a negative control for background staining. All samples were processed on a BOND-III autostainer (Leica Microsystems, Buffalo Grove, Illinois, USA) or IntelliPATH automated stainer (Biocare Medical, Pacheco, California, USA) at ambient temperature according to the manufacturer’s instructions. Each IHC run included a B7-H4 positive control (HEK293T B7-H4-overexpressing cell pellet) and a B7-H4 negative control (HEK293T parental cell pellet) to confirm the anti-B7-H4 antibody clone D1M8I was sensitive and specific for B7-H4. Images were captured using a slide scanner (Leica, Aperio AT2, Buffalo Grove, Illinois, USA) and glass slides and/or digital images were evaluated and scored by a pathologist as follows: Intensity: 0=none, 1=weak, 2=moderate, 3=strong. Membrane and/or apical tumor cell staining on tumor cores or full tumor sections was scored in figure 1A as indicated; total tumor cell staining (membrane, apical, or cytoplasmic) on full tumor sections was scored in figure 1B. For prevalence calculations, tumors were considered positive if staining was observed on greater than 25% of tumor cells. Tumors scored with intensities “1–2” or “2–3” were plotted as the lower intensity score number.
B7-H4 expression is elevated on multiple solid tumor types, including breast, ovarian, and endometrial tumors. (A) Quantification of membrane and/or apical B7-H4 staining on various tumor cores or full tumor sections as indicated. (B) Quantification of total (membrane, apical, or cytoplasmic) B7-H4 staining on full tumor sections as indicated. (C, D) Images of formalin-fixed, paraffin-embedded triple-negative breast cancer (TNBC, membrane B7-H4 staining on 100% of tumor cells with overall intensity of 3+) (C) and endometrioid carcinoma (apical cytoplasmic and some true membrane B7-H4 staining on 100% of tumor cells with overall intensity of 3+) tumors (D) stained for B7-H4 by immunohistochemistry using monoclonal antibody clone D1M8I. Tumor tissue in panels A and B were stained on a BOND-III autostainer and IntelliPATH automated stainer, respectively, and scored by two different pathologists as follows: Intensity: 0=none, 1=weak, 2=moderate, 3=strong. For prevalence calculations, tumors were considered positive if staining was observed on greater than 25% of tumor cells. Tumors scored with intensities “1–2” or “2–3” were plotted as the lower intensity score number. BC, breast cancer; HER2+, human epidermal growth factor receptor 2-positive; HR+, hormone receptor-positive; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer; TMA, tissue microarray.
In vivo activity in xenograft models of breast and ovarian cancer
MX-1 and MDA-MB-468 xenograft models
All in vivo experiments were approved by the Institutional Animal Cancer and Use Committees (IACUC) of Seagen under protocol number SGE-029 and the ARRIVE reporting guidelines were used.29 Specific pathogen-free (SPF) mice were purchased from Taconic or Jax and housed in an SPF barrier facility. Tumor cell lines were tested for select pathogens and were negative. Female severe combined immunodeficient (SCID, C.B-Igh-1b/IcrTac-Prkdcscid, Taconic, San Diego, California, USA) mice were implanted with 5×105 MX-1 tumor cells in 25% Matrigel HC subcutaneously. Once tumor volumes reached 100 mm3, mice were randomized into treatment groups and dosed with 3 mg/kg of ADC every 7 days for three total doses. Tumor volumes were measured twice per week, and animals were euthanized when tumor volume reached 700–1000 mm3. Stock concentrations of ADC were diluted to a desired concentration (with 0.01% Tween 20 in phosphate-buffered saline (PBS)) and injected intraperitoneally into each treatment group.
Female NOD scid gamma (NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/Szj, Jax, Bar Harbor, Maine, USA) mice were implanted with 1×106 MDA-MB-468 cells in 25% Matrigel HC subcutaneously. Once tumor volumes reached 100 mm3, mice were randomized into treatment groups and dosed with 0.3, 1, or 3 mg/kg every 7 days for three total doses. Twenty-four hours prior to receiving each ADC dose, each animal was treated with 10 mg/kg human intravenous immunoglobulin (hIVIG). Tumor volumes were measured twice per week, and animals were euthanized when tumor volume reached 700–1000 mm3. Stock concentrations of ADC were diluted to a desired concentration (with 0.01% Tween 20 in PBS) and injected intraperitoneally into each treatment group.
Patient-derived xenograft models
All in vivo experiments were performed according to the guidelines of the IACUC of Champions Oncology (Hackensack, New Jersey, USA) under protocol number 2020-TOS-001. Fragments from Champions TumorGraft models representing human triple-negative breast cancer (TNBC), ovarian cancer, or hormone receptor-positive (HR+) breast cancer were bilaterally implanted into stock NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG, Taconic; TNBC) or Hsd:Athymic Nude-Foxn1nu (Nude, Envigo; HR+ breast cancer and ovarian cancer) mice. Once tumor volumes reached 1000–1500 mm³, they were harvested, and the tumor fragments were implanted subcutaneously in the left flank of the female study mice. When the tumor volume reached approximately 150–300 mm³, animals were matched by tumor size and assigned into control (untreated) or treatment groups. Mice in the treatment group were treated with 3 mg/kg of ADC intravenously every 7 days for three total doses. NOG mice implanted with TNBC patient-derived xenograft (PDX) tumors were treated with 10 mg/kg hIVIG intravenously 24 hours prior to ADC dosing. Tumor size and body weight were measured twice per week, and the study was terminated when tumors in the control group reached 1500 mm³ or up to Day 28, whichever occurred first.
SGN-B7H4V antitumor activity in an immunocompetent murine tumor model alone and in combination with an anti-PD-1 agent
Renca efficacy studies
All in vivo experiments were approved by the IACUC of Seagen under protocol number SGE-029 and the ARRIVE reporting guidelines were used.29 SPF mice were purchased from Envigo and housed in an SPF barrier facility. Tumor cell lines were tested for select pathogens and were negative. Murine B7-H4-expressing Renca cells (parental Renca cell line: ATCC-CRL-2947) were cultured in RPMI-1640 medium (American Type Culture Collection (ATCC), Manassas, Virginia, USA) with 10% heat-inactivated fetal bovine serum, minimum essential medium (MEM) non-essential amino acids (1×), sodium pyruvate (1 mM), and L-glutamine (2 mM). Renca cancer cells were implanted (2×106 cells in 200 µL 25% Matrigel) subcutaneously into Balb/c female mice (BALB/cAnNHsd, Envigo, Indianapolis, Indiana, USA). Once tumor volumes reached approximately 100 mm3, mice were randomized into treatment groups and treated intraperitoneally with 3 weekly doses of 3 mg/kg SGN-B7H4V, non-binding control ADC, B7H41001 mAb, anti-PD-1 mAb (clone 29F.1A12, Bio X Cell #BP0273), or isotype control mAb (clone 2A3, Bio X Cell #BP0089). ADCs and mAbs with a murine IgG2a (mIgG2a) Fc backbone were used to avoid antidrug antibody (ADA) responses that can occur on repeat treatment with human IgG1 antibodies in immunocompetent mice.
Tumor-bearing mice that achieved complete tumor regression following ADC treatment were “re-challenged” with parental (ie, not B7-H4-expressing) Renca tumor cells; Renca cancer cells were implanted (2×106 cells in 200 µL 25% Matrigel in RPMI 1640 medium) subcutaneously into the opposite flank of Balb/c mice.
In some experiments, mice were randomized into treatment groups once tumor volumes reached approximately 250 mm3 and treated intraperitoneally with a single 3 mg/kg dose of ADC or mAb. Six to 7 days following treatment, tumors were harvested at necropsy, cut in half, and processed for RNA-sequencing (RNAseq) (frozen at 80°C) or FFPE for immunohistochemical analysis.
Analysis of in vivo antitumor activity in murine tumor models
Per cent tumor growth inhibition (%TGI) was calculated on the day at which animals in the control group were terminated by the Jackson Labs method, defined as:
Tumor growth was assessed by the metric area under the curve (AUC), “AUC.3”, based on Guo et al’s best practices for reporting preclinical data, which reflects area under a continuous growth curve and accounts for time on study30:
To determine whether a treatment elicited statistically significant antitumor activity compared with the control group, a one-way analysis of variance test was performed followed by Tukey’s post hoc multiple comparisons test to compare the AUC.3 values from each treatment group to the untreated control group for each model.
Tumors were considered to have a “complete response” (CR) if the tumor volumes were <60 mm3 for the last 7 days of measurement.
Results
B7-H4 expression is elevated on multiple solid tumor types, including breast, ovarian, and endometrial tumors
B7-H4 expression on multiple solid tumor samples was evaluated by IHC using an anti-B7-H4 mAb that selectively stains B7-H4 expressing cells (online supplemental figure 1). Tumor cores on TMAs were stained and scored as part of an initial screen for B7-H4 expression (figure 1A). Given that TMAs are often subject to variation due to multiple preanalytical factors (fixation time, location of core within the tumor, etc), full tumor tissue sections were subsequently stained and scored for B7-H4 prevalence calculations (figure 1A,B). B7-H4 expression was detected across a range of solid tumor types, with representative images of B7-H4 staining on a TNBC and endometrioid carcinoma full tumor section shown in figure 1. Notably, approximately 50%–80% of ovarian, breast, and endometroid tumors were found to express B7-H4. Complete prevalence and staining intensity of B7-H4 are summarized in figure 1A,B. These data support the notion that B7-H4 expression is upregulated in tumors, in particular breast, ovarian, and endometrial tumors. In contrast to elevated B7-H4 expression on tumor cells, limited B7-H4 expression was observed on normal tissues (online supplemental table 1) and immune cells (online supplemental figure 2).
Supplemental material
Supplemental material
Supplemental material
SGN-B7H4V is internalized following binding to B7-H4 and kills tumor cells via MMAE-mediated direct cytotoxicity
SGN-B7H4V comprises the B7-H4-directed human mAb (B7H41001 mAb, see supplemental results and online supplemental table 3) conjugated to the cytotoxic payload MMAE via a protease-cleavable mc-vc linker. Automated immunofluorescence was used to visualize internalization properties of the B7H41001 antibody component in a cell-based assay. MX-1 TNBC cells were incubated with B7H41001 mAb or non-binding control mAb conjugated to a quenched fluorophore using the same mc-vc linker used in SGN-B7H4V. Unquenched fluorescence was monitored as a proxy for internalization and mc-vc linker cleavage. The fluorescent signal in this assay increased with an apparent half-time of approximately 4.9 hours, suggesting quick internalization of SGN-B7H4V (figure 2A and online supplemental figure 3). Importantly, internalization was dependent on binding to B7-H4 as minimal fluorescence was detected when cells were incubated with the non-binding control mAb-quenched fluorophore conjugate.
Supplemental material
SGN-B7H4V is internalized following binding to B7-H4 and kills tumor cells via MMAE-mediated direct cytotoxicity. (A) Quantification of unquenched fluorescence over time following incubation of MX-1 cells with B7H41001 mAb or non-binding control mAb conjugated to a quenched fluorophore using the same maleimidocaproyl valine-citrulline linker as in SGN-B7H4V. (B) Summary of mean half maximal cytotoxic concentration values (defined as the concentration of ADC required to reduce cell viability to 50%) of MX-1, SKBR3, MDA-MB-468, and MDA-MB-231 tumor cell spheroids 96 hours following incubation with SGN-B7H4V or a non-binding control ADC. B7-H4 surface expression levels on each cell line as determined by quantitative flow cytometry are indicated in the top panel. (C) Quantification of cell viability of MX-1 tumor cell spheroids 96 hours following incubation with the indicated doses of SGN-B7H4V or a non-binding control ADC. ADC, antibody–drug conjugate; mAb, monoclonal antibody; MMAE, monomethyl auristatin E; RFU, relative fluorescence units.
The ability of SGN-B7H4V to elicit cytotoxicity in an in vitro assay was determined using three B7-H4-expressing breast cancer cell lines (SKBR3, MX-1, and MDA-MB-468) and one control non-B7-H4-expressing breast cancer cell line (MDA-MB-231) grown in spheroid conditions. All three of the B7-H4-expressing cell lines exhibited sensitivity to SGN-B7H4V, but not to the non-binding control ADC, with mean half maximal cytotoxic effective concentration values ranging from 6 ng/mL to 77 ng/mL (figure 2B,C). SGN-B7H4V demonstrated more potent cytotoxic activity on the cell lines with higher B7-H4 surface expression levels as determined by quantitative flow cytometry (ie, MX-1 and SKBR3 compared with MDA-MB-468), suggesting B7-H4 expression level may be correlated with the potency of in vitro cell killing by SGN-B7H4V. These data demonstrate SGN-B7H4V internalizes quickly into B7-H4-expressing cells following binding, releases MMAE, and kills tumor cells via MMAE-mediated direct cytotoxicity.
SGN-B7H4V mediates ADCC and ADCP in vitro
In addition to direct cytotoxicity elicited by MMAE, antibodies directed to cell surface antigens can elicit direct killing of antibody-coated cells via engagement of the antibody Fc backbone with activating Fcγ receptors on natural killer (NK) cells (ADCC) or macrophages (ADCP).31–33 The ability of SGN-B7H4V, B7H41001 mAb, a non-binding control ADC, and/or a non-binding control mAb to induce ADCC and ADCP in vitro was evaluated using human B7-H4-expressing breast cancer cell lines and primary NK cells or monocytes/macrophages. Both SGN-B7H4V and B7H41001 mAb, but not the non-binding controls, elicited comparable ADCC (figure 3A) and ADCP (figure 3B) responses in vitro. This indicates that, in vitro, SGN-B7H4V may also drive tumor cell killing via the antibody-mediated effector functions ADCC and ADCP and, moreover, that conjugation of MMAE to B7H41001 mAb does not meaningfully interfere with the ability of the Fc backbone to engage Fcγ receptors and drive these antibody-mediated effector functions. While these data indicate in vitro MOAs the contribution to total antitumor activity is hard to delineate.
SGN-B7H4V kills tumor cells by ADCC and ADCP in vitro. (A) Quantification of tumor cell lysis 4 hours after incubation of purified human natural killer cells with MX-1 cells treated with SGN-B7H4V, B7H41001 mAb, a non-binding control ADC, or a non-binding mAb. (B) Quantification of tumor cell phagocytosis (plotted as the PKH26 gMFI on CD14+/CD45+ cells) following overnight incubation of monocytes/macrophages with PKH26-labeled SKBR3 cells treated with SGN-B7H4V, B7H41001 mAb, or a non-binding mAb. ADC, antibody–drug conjugate; ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; gMFI, geometric mean fluorescence intensity; mAb, monoclonal antibody.
SGN-B7H4V demonstrates robust activity in vivo in multiple xenograft models of breast and ovarian cancer
To determine if the robust activity of SGN-B7H4V observed in vitro translated to an in vivo setting, the antitumor activity of SGN-B7H4V was evaluated in two cell-line derived xenograft (CDX) models of TNBC as well as three PDX models of TNBC and ovarian cancer. MDA-MB-468 CDX tumor-bearing NSG mice were treated with SGN-B7H4V, B7H41001 mAb, or non-binding control ADC at 0.3, 1, and/or 3 mg/kg for 3 weekly doses when tumors were approximately 100 mm3. SGN-B7H4V elicited antitumor activity at the 1 and 3 mg/kg dose levels (mean TGI: 62.2% and 90.7%, respectively; mean AUC.3: 0.012 (p=0.0786) and −0.010 (p<0.0001), respectively), though activity was not statistically significantly different at the 1 mg/kg dose level compared with untreated tumors. In contrast, treatment with 0.3 mg/kg SGN-B7H4V, 3 mg/kg B7H41001 mAb, or 3 mg/kg non-binding control ADC had minimal antitumor activity (figure 4A and online supplemental table 4).
SGN-B7H4V demonstrates robust activity in vivo in multiple xenograft models of breast and ovarian cancer. (A,B) Mean tumor volume over time of MDA-MB-468 tumor-bearing NSG mice following treatment. (B) Mean tumor volume over time of MX-1 tumor-bearing nude mice following treatment. (C) Tumor volume over time of PDX tumor-bearing nude mice following treatment; tumor volume for individual mice (SGN-B7H4V) or mean tumor volume (untreated and non-binding control ADC) is plotted. Representative images of IHC staining for B7-H4 on untreated xenograft tumors is shown in the bottom of panels (A–C). PDX model metadata are shown in panel (D). All CDX and PDX tumor-bearing mice were treated with 3 weekly doses of SGN-B7H4V, B7H41001 mAb, and/or non-binding control ADC as indicated. Statistical analysis of antitumor activity was performed as described in online supplemental table 4. ADC, antibody–drug conjugate; CDX, cell-line derived xenograft; IHC, immunohistochemistry; mAb, monoclonal antibody; mRNA, messenger RNA; PDX, patient-derived xenograft; TNBC, triple-negative breast cancer.
Similarly, MX-1 CDX tumor-bearing nude mice were treated with SGN-B7H4V or non-binding control ADC (3 mg/kg for 3 weekly doses) when tumors were approximately 100 mm3. SGN-B7H4V elicited antitumor activity (mean TGI: 96.5%; mean AUC.3: −0.042 (p=0.0009)), with sustained regression in four of the five mice. In contrast, the non-binding control ADC had minimal antitumor activity (figure 4B and online supplemental table 4).
To confirm the potential of SGN-B7H4V to elicit anticancer activity in human tumor samples, PDX tumor fragment-bearing NOG (TNBC) or nude mice were treated with SGN-B7H4V or non-binding control ADC (3 mg/kg for 3 weekly doses) when tumors were 150–300 mm3. In a PDX model of TNBC with heterogeneous B7-H4 staining, SGN-B7H4V elicited tumor regression (mean TGI: 97.5%; mean AUC.3: −0.083 (p=0.0003); figure 4C, left panel and figure 4D). Additionally, SGN-B7H4V elicited tumor regression in a PDX model of ovarian cancer with uniformly high B7-H4 staining (mean TGI: 96.1%; mean AUC.3: −0.019 (p=0.0008); figure 4C, middle panel and figure 4D) and tumor growth delay in a heavily pretreated PDX model of ovarian cancer with heterogeneous B7-H4 staining (mean TGI 89.1%: mean AUC.3: −0.007 (p=0.0014); figure 4C, right panel, figure 4D, and online supplemental table 4). Altogether, SGN-B7H4V demonstrates strong antitumor activity in xenograft tumor models, including those with heterogeneous expression of B7-H4 consistent with the bystander effect ascribed to vedotin ADCs.34–37
SGN-B7H4V demonstrates robust antitumor activity accompanied by immunomodulatory changes in vivo in an immunocompetent murine tumor model
In addition to tumor cell killing, SGN-B7H4V may drive immunomodulatory effects through MMAE-mediated ICD and/or internalization of the B7-H4/ADC complex from the surface of cells in the tumor microenvironment (TME). Therefore, we sought to evaluate the antitumor activity and subsequent immunomodulatory changes in tumors following treatment with SGN-B7H4V. Immunodeficient xenograft tumor models lack T cells and are limited to evaluation of innate immune cell activity; therefore, to evaluate the antitumor and immunomodulatory activity of SGN-B7H4V in an immunocompetent setting, a murine B7-H4 (mB7-H4)-expressing Renca syngeneic tumor model was developed (online supplemental figure 4). ADCs and mAbs with an mIgG2a Fc backbone were used to avoid deleterious ADA responses against human antibodies that may occur in immunocompetent mice.
Supplemental material
mB7-H4-Renca tumor-bearing mice were treated with 3 weekly doses of 3 mg/kg SGN-B7H4V, B7H41001 mAb (which demonstrates comparable binding to murine and human B7-H4, see supplemental results), or non-binding control ADC when tumor volumes reached 100 mm3. Treatment with SGN-B7H4V elicited robust antitumor activity (mean TGI: 91.9%, mean AUC.3: −0.016 (p=0.0051)), while in contrast the non-binding control ADC elicited modest tumor growth delay (figure 5A and online supplemental table 4). Neither the fucosylated nor non-fucosylated (Fc effector function-enhanced) B7H41001 mAbs elicited significant antitumor activity (figure 5A and online supplemental table 4), indicating that Fc effector function alone is not sufficient to account for the antitumor activity seen in mice treated with SGN-B7H4V.
SGN-B7H4V demonstrates robust antitumor activity accompanied by immunomodulatory changes in vivo in an immunocompetent murine tumor model. (A) Mean tumor volume over time of mB7-H4-Renca tumor-bearing Balb/c mice following treatment with SGN-B7H4V, non-binding control ADC, B7H41001 mAb, or the non-fucosylated B7H41001 mAb (3 mg/kg, 3 weekly doses). (B) Immunohistochemical staining of F4/80 and CD11c in tumor sections 6–7 days following treatment with a single 3 mg/kg dose of SGN-B7H4V, non-binding control ADC, or B7H41001 mAb (top panel, representative IHC images; bottom panel, quantification of the % positive tissue in tumor bed). (C) Quantification of the following transcripts in tumors treated as in (B): Cd86 and Icosl (encode the co-stimulatory molecules CD86 and ICOS-L), H2-Aa and H2-eb1 (encode major histocompatibility complex class II molecules), as well as Itgax (encodes CD11c) and Batf3 (encodes BatF3, which is involved in antigen cross-presentation). (D) Immunohistochemical staining of CD3, CD4, CD8, and PD-1 in tumors treated as in (B) (left panel, representative IHC images; right panel, quantification of the % positive cells in tumor bed). (E) Quantification of the following transcripts in tumors treated as in (B): Cd3e, Cd4, Cd8a, Pdcd1 (encodes PD-1) and Icos (encodes ICOS). Statistical analysis for IHC was performed using an unpaired t-test with Holm-Sidak’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001. Statistical differences for RNA-sequencing were determined based on the raw read counts between groups of samples using DESeq2 V.1.28.1. Adjusted p value: **p<0.01; ***p<0.001. ADC, antibody–drug conjugate; IHC, immunohistochemistry; mAb, monoclonal antibody; PD-1, programmed cell death-1; TPM, transcripts per million reads; ICOS, inducible costimulator.
Vedotin ADCs have been described to elicit hallmarks of ICD, including the exposure of calreticulin on the cell surface and extracellular release of ATP and high-mobility group box 1.25 26 38–40 These hallmarks serve as damage-associated molecular patterns (DAMPs) that may recruit and activate innate immune cells, resulting in antitumor activity.25 Consistent with other vedotin ADCs, SGN-B7H4V induces hallmarks of ICD in vitro (online supplemental figure 5). Moreover, SGN-B7H4V binds and internalizes the B7-H4/ADC complex. This may, in combination with SGN-B7H4V-induced ICD, enhance T-cell activation. Thus, mB7-H4-Renca tumors grown in immunocompetent hosts were evaluated to explore whether the robust antitumor activity described above was accompanied by immunomodulatory changes in the TME in vivo.
Supplemental material
mB7-H4-Renca-tumor bearing mice were treated with a single 3 mg/kg dose of SGN-B7H4V, non-binding control ADC, or B7H41001 mAb. Tumors were harvested 6–7 days after treatment, cut in half, and evaluated using RNAseq or IHC. RNAseq analysis of SGN-B7H4V-treated tumors revealed a significant increase in transcripts encoding cytokines and type I interferon response genes (online supplemental figure 6A) following treatment with SGN-B7H4V. Given that these cytokines may promote immune cell activation and recruitment to tumors, we next evaluated tumors for the presence of immune cells. Quantification of stained tumor sections revealed an increase in the proportion of CD11c+ dendritic cells and F4/80+ macrophages in SGN-B7H4V-treated tumors (figure 5B). This was corroborated by RNAseq analysis, which revealed an increase in Itgax (which encodes CD11c), Batf3 (which encodes BatF3, a transcription factor associated with antigen cross-presentation), H2-Aa and H2-eb1 (which encode major histocompatibility complex (MHC) class II molecules), and Cd86 and Icosl (which encode co-stimulatory molecules) transcripts following treatment with SGN-B7H4V (figure 5C). This suggests that SGN-B7H4V can promote recruitment of innate antigen-presenting cells to tumors as well as upregulation of genes associated with antigen presentation to T cells, including MHC class II molecules and multiple co-stimulatory molecules. Quantification of stained tumor sections also revealed an increase in the proportion of CD3+, CD4+, and CD8+T cells as well as cells expressing PD-1, the receptor for PD-L1 that is upregulated on newly activated T cells, in SGN-B7H4V-treated tumors (figure 5D). The morphology of the PD-1+ cells was consistent with lymphocytes: the cells are small, round to polygonal, with high nuclear to cytoplasmic ratio and membranous PD-1 signal. This was in agreement with the RNAseq analysis, which revealed an increase in Cd3e, Cd4, Cd8a (which encode the T-cell markers CD3, CD4, and CD8, respectively) as well as Pdcd1 and Icos (which encode the early T-cell activation markers PD-1 and inducible costimulator (ICOS)) following treatment with SGN-B7H4V (figure 5E). Altogether, these combined data are supporting evidence that SGN-B7H4V can promote recruitment of innate and adaptive T cells to tumors and upregulation of genes associated with antigen presentation and early T-cell activation. The phenotype of immune cells in the TME should be further explored in future studies using dual chromogenic IHC, immunofluorescence, in situ hybridization, and/or single-cell RNAseq approaches.
Supplemental material
Response to anti-PD-(L)1 agents in the clinic has been associated with expression of PD-L1 and/or expression of a “T-cell–inflamed” gene signature.41 Quantification of stained tumor sections also revealed an increase in the proportion of PD-L1+ cells following treatment with SGN-B7H4V (online supplemental figure 7). RNAseq analysis revealed an increase in multiple murine genes associated with a “T-cell–inflamed” gene signature (online supplemental figure 6B). These data support the notion that MMAE-mediated ICD induced by SGN-B7H4V elicits robust recruitment of both innate antigen-presenting cells as well as cytotoxic T cells to tumors, in addition to direct cell killing. These data indicate that SGN-B7H4V may elicit immunomodulatory changes to the TME that may promote responsiveness to anti-PD-(L)1 agents.
Supplemental material
SGN-B7H4V combines with an anti-PD-1 agent and induces long-term immune memory
The activity of SGN-B7H4V was evaluated in combination with an anti-PD-1 agent in the mB7-H4-Renca immunocompetent tumor model. Given the robust antitumor activity observed following treatment with 3 mg/kg SGN-B7H4V, a subtherapeutic dose (1 mg/kg) was used in combination studies. mB7-H4-Renca tumor-bearing mice were treated with 3 weekly doses of 1 mg/kg SGN-B7H4V or non-binding control ADC alone or in combination with 0.3 mg/kg anti-PD-1 antibody when tumor volumes reached 100 mm3. Treatment with SGN-B7H4V in combination with the anti-PD-1 mAb led to enhanced survival with more CRs (12/30 (40%)) observed compared with either treatment alone (1/20 (5%)) or SGN-B7H4V in combination with a rat isotype control mAb (2/30 (7%), figure 6A,B, online supplemental table 4).
SGN-B7H4V combines with an anti-PD-1 agent and induces long-term immune memory. (A) Per cent survival of mB7-H4-Renca tumor-bearing mice treated with 3 weekly doses of 1 mg/kg ADC and 0.3 mg/kg anti-PD-1 antibody alone or in combination. (B) Individual tumor volume of tumor-bearing mice that were treated as in (A). (C) Mice with complete responses were rechallenged with parental (non-B7-H4-expressing) Renca tumor cells. Seven out of 12 mice (58%) previously treated with SGN-B7H4V in combination with an anti-PD-1 mAb were protected, compared with only 3 out of 10 mice (30%) treated with SGN-B7H4V alone. Statistical analysis of overall survival (A) was performed using a log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, ***p<0.001. See online supplemental table 3 for statistical analysis of tumor growth described in panel B. Anti-PD-1, anti-programmed cell death-1; CR, complete response; mAb, monoclonal antibody; Q1W, weekly.
Finally, tumor rechallenge studies were conducted with mice that demonstrated CRs to evaluate the ability of SGN-B7H4V to elicit durable immune memory when administered in combination with an anti-PD-1 agent. Mice treated with 3 mg/kg SGN-B7H4V (figure 5A) or 1 mg/kg SGN-B7H4V in combination with 0.3 mg/kg anti-PD-1 mAb (figure 6A) that achieved durable tumor regression were rechallenged with parental (non-B7-H4-expressing) Renca tumor cells. Seven out of 12 mice (58%) previously treated with SGN-B7H4V in combination with an anti-PD-1 mAb were protected from tumor rechallenge, compared with protection in 3 out of 10 mice (30%) treated with SGN-B7H4V alone (figure 6C). This highlights the ability of SGN-B7H4V to induce durable immune memory as monotherapy and more robustly when administered in combination with an anti-PD-1 agent.
Discussion/conclusion
SGN-B7H4V is a novel B7-H4-directed investigational vedotin ADC currently being evaluated in a phase 1 clinical trial for patients with advanced solid tumors (SGNB7H4V-001, NCT05194072).5
Here, we found B7-H4 expression was elevated on multiple solid tumors tested, including breast, ovarian, endometrial, NSCLC, and cholangiocarcinoma tumors while expression was limited in normal tissue. In contrast to other B7 family members (eg, B7-H3), B7-H4 expression was very low or absent on immune cells, reducing the potential for deleterious molecular target-driven immune cell toxicity.42 Indeed, we observed robust recruitment of immune cells to tumors—rather than evidence of immunosuppressive effects—elicited by SGN-B7H4V in an immunocompetent murine tumor model (figure 5). Altogether, the elevated expression pattern of B7-H4 in tumors (where it may promote immune evasion) versus healthy tissue makes it an ideal molecular target for ADCs.
Vedotin ADCs are directed to molecular targets that are prevalent on the surface of target cells, and once bound to the target cell surface, are internalized and trafficked to the lysosome. Following vedotin ADC internalization, lysosomal proteases cleave the mc-vc linker and release MMAE into the cytoplasm, where it induces apoptosis by binding and disrupting the microtubule network.24 26 MMAE-mediated direct cytotoxicity is believed to be an important component of the multifaceted MOA of SGN-B7H4V. We therefore evaluated the internalization properties as well as the cytotoxic activity of SGN-B7H4V in vitro. We found that B7H41001 mAb is internalized and SGN-B7H4V exerts cytotoxic activity on B7-H4-expressing cells, suggesting SGN-B7H4V can deliver the cytotoxic payload MMAE and kill cells that express B7-H4. In addition to direct MMAE-mediated cytotoxicity, we found that, in vitro, SGN-B7H4V can kill tumor cells via ADCC and ADCP. However, the contribution of these processes to the antitumor activity observed in vivo is unclear. While it may be the case that activity could vary across model systems, B7H1001 mAb elicited no significant antitumor activity in our study (figures 4A and 5A), even when used as a non-fucosylated Fc-effector-function-enhanced version.
Consistent with the significant activity observed in vitro, SGN-B7H4V demonstrated robust antitumor activity in vivo in multiple xenograft models of breast and ovarian cancer including two CDX models of human TNBC as well as three PDX models of TNBC or ovarian cancer. Activity was observed across a range of B7-H4 expression levels, including tumors with heterogeneous B7-H4 staining, and in both treatment-naïve and a heavily pretreated metastatic ovarian tumor. The activity of SGN-B7H4V across a range of B7-H4 expression levels is consistent with multiple potential mechanisms of action in vivo, including direct cytotoxicity as well as the bystander effect, wherein the cell-permeable MMAE payload diffuses across cell membranes to cause apoptosis in adjacent tumor cells independent of their molecular target expression level (figure 4).34 40
In addition to direct MMAE-mediated cytotoxicity and the bystander effect, ICD may be induced following MMAE-mediated killing by vedotin ADCs. Cells that undergo apoptosis as a result of exposure to MMAE release DAMPs, which may prompt recruitment and activation of immune cells to tumors to drive antitumor immunity.25 Vedotin ADCs, including SGN-B7H4V, have been shown to elicit hallmarks of ICD in vitro and drive immunomodulatory changes in vivo in preclinical tumor models (online supplemental figure 5).25 26 40
For the first time, in this study, the ability of SGN-B7H4V to elicit immune changes in tumors in an immunocompetent tumor model was evaluated. SGN-B7H4V demonstrated robust antitumor activity in vivo, accompanied by immunomodulatory changes, in an immunocompetent syngeneic model. Specifically, SGN-B7H4V led to recruitment of innate antigen-presenting cells and upregulation of genes associated with antigen presentation to T cells as well as recruitment of adaptive T cells and upregulation of early T-cell activation markers, including PD-1 and ICOS. Moreover, SGN-B7H4V led to immunomodulatory changes (eg, upregulation of PD-L1) that have been associated clinically with response to anti-PD-(L)1 agents, providing a strong rationale for combination with other immunomodulatory therapeutic modalities, particularly anti-PD-(L)1 agents.28 Indeed, the combination of SGN-B7H4V with an anti-PD-1 agent led to improved antitumor activity compared with either treatment alone, with combination therapy driving improved overall survival and durable immune memory (figure 6). This durable antitumor immunity is consistent with a model in which SGN-B7H4V-mediated induction of ICD drives local immune cell activation and recruitment to tumors that, when combined with anti-PD-1 agents to remove T-cell checkpoints, drives optimal long-term antitumor immunity.25 Consistent with this model, the complementary combination of vedotin ADCs with immune checkpoint inhibitors has been demonstrated by clinically meaningful responses observed when brentuximab vedotin, enfortumab vedotin, or tisotumab vedotin are paired with anti-PD-1 agents.28 43 44 The data presented here suggest SGN-B7H4V has the potential to show combinatory antitumor activity with PD-1 inhibition.
SGN-B7H4V, in which MMAE is stochastically conjugated to native cysteines on a B7-H4-directed mAb (DAR ~4), is the first B7-H4-directed vedotin ADC in clinical development. Multiple additional B7-H4-directed ADCs have been evaluated preclinically and/or are currently being evaluated in phase 1 clinical trials. These include a B7-H4 THIOMAB ADC that was evaluated preclinically in which MMAE is site-specifically conjugated via the same peptide cleavable mc-vc linker as SGN-B7H4V to a B7-H4-directed THIOMAB (DAR=2).9 Other B7-H4-directed ADCs in clinical development use different drug-linker payload systems including a topoisomerase I inhibitor payload (DAR=8) and a site-specifically conjugated microtubule-disrupting auristatin payload (DAR=6).45 46 SGN-B7H4V is distinct from other B7-H4-directed ADCs in that it leverages the clinically validated vedotin drug-linker payload technology platform.
In conclusion, these results strengthen our understanding of B7-H4 expression on solid tumors and demonstrate limited B7-H4 expression on normal tissue. In addition, these data provide direct evidence for the multimodal MOA of SGN-B7H4V, demonstrate its antitumor activity in in vivo xenograft models, and support the continued investigation of the immunomodulatory activity of SGN-B7H4V and potential of SGN-B7H4V to combine with immune checkpoint inhibitors.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
Ethics statements
Patient consent for publication
Ethics approval
Champions Oncology acquires patient tumor tissue from multiple sources in order to develop PDX tumor models. All patients are consented using an ethics approved consent form.
Acknowledgments
The authors would like to thank Rogely Waite Boyce DVM, PhD, DACVP, for scoring of IHC stained tumor tissue. Medical writing support, including assisting authors with the development of the outline and initial draft and incorporation of comments, was provided by Joanna Lamprou, PharmD, and Anastasija Pesevska, PharmD, and editorial support, including referencing, figure preparation, formatting, proofreading, and submission was provided by Travis Taylor, BA, all of Scion of London, supported by Seagen Inc. according to Good Publication Practice guidelines (Link). The Sponsor was involved in the study design, collection, analysis and interpretation of data, as well as data checking of information provided in the manuscript. However, ultimate responsibility for opinions, conclusions, and data interpretation lies with the authors.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors EG designed all experiments, supervised and guided the analysis, andwrote the manuscript. MU performed in vivo experiments. EG, EJH, AE, PY, SW, KSp, and KSn performed in vitro experiments. DS, SA, L-YH, EJH, and KG performed all IHC staining. KH, PMT, EST, DM, and MP analyzed IHC staining. JJG and RT performed bioinformatic analysis of the RNAseq data. AJS, CF, JPS, NN, and SJG provided critical intellectual input. All authors reviewed and edited the manuscript. EG is responsible for the overall content of the manuscript as the guarantor.
Funding This work was funded by Seagen Inc.
Competing interests EG, MU, AE, PY, DS, KH, SA, L-YH, JH, KG, PMT, EST, JJG, RT, SW, KS, EJH, KSn, DM, MP, AJS, CF, JPS, NN, and SJG were employees of and had equity ownership in Seagen Inc. at the time of this work.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.