Correspondence to Dr Thorbald van Hall; [email protected]
WHAT IS ALREADY KNOWN ON THIS TOPIC
CD3 bispecific antibodies (CD3 bsAbs) have emerged as an established treatment modality in the treatment of hematological malignancies and, more recently, also solid tumors. Antigen loss is one of the dominant escape mechanisms in the context of CD3 bsAb treatment and impedes long-term therapeutic efficacy. The few studies that investigated the generation of tumor-specific responses, crucial to prevent recurrences, showed contradicting results.
WHAT THIS STUDY ADDS
We demonstrate that a combination of CD3 bsAbs with tumor-specific vaccination improves survival against primary tumors and, importantly, also induces durable antitumor responses that confer protection from rechallenge in immunologically “hot” as well as “cold” solid tumor models.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY
This study provides a strong rationale for the combination of CD3 bsAbs and tumor-specific vaccination. In light of recent clinical successes of the therapeutic messenger RNA-based cancer vaccines, we advocate further translational implementation of our combinatorial concept.
Background
Over the last decade, CD3 bispecific antibody (CD3 bsAb) or “T-cell engager” therapy has become an established treatment modality for the treatment of certain B-cell malignancies. Treatment with CD3 bsAbs achieved its breakthrough in the clinic with blinatumomab (CD3xCD19), which demonstrated response rates up to 90% in relapsed or refractory acute lymphoblastic B-cell leukemia.1 Following this initial success, six other CD3 bsAbs targeting B-cell antigens, one targeting delta-like ligand 3 for small cell lung cancer and one CD3 bispecific molecule targeting a peptide/major histocompatibility complex expressed in uveal melanoma have been Food and Drug Administration (FDA)-approved.2–8 As already suggested by the difference in the number of FDA-approved bsAbs, clinical development of the CD3 bsAb is lagging behind in solid tumor indications, where safety and efficacy are major obstacles.9
BsAbs act by crosslinking a tumor-associated antigen expressed on the surface of tumor cells to CD3ε on the surface of T cells, resulting in the formation of an immunological synapse, followed by T-cell activation and tumor cell lysis.9 As binding to CD3ε is independent of T-cell specificity, an important advantage of CD3 bsAbs is the ability to engage all T cells in the tumor microenvironment (TME). Although these therapeutics activate intratumoral T cells, it is uncertain if this therapy invigorates tumor-specific T cells or leads to the induction of de novo antitumor T cells via antigen spread. According to the cancer-immunity cycle paradigm, initial cancer cell kill might lead to the presentation of tumor antigens by professional antigen-presenting cells (APCs) followed by ignition of tumor-specific T-cell responses.10 The presence of systemic tumor-specific T cells is essential to fend off recurrences of tumor cells or (dormant) metastases that were not destroyed during the therapeutic intervention. Additionally, the induction of such responses could help to overcome tumor heterogeneity or resistance due to loss of surface expression of the targeted epitope, which are frequently reported causes for relapse after CD3 bsAb therapy in the clinic for hematological malignancies,11–16 as will likely also be the case for solid tumors once CD3 bsAb therapy for these cancer indications is successfully applied in the clinic.
Thus far, most studies involving CD3 bsAbs focus on specificity and therapeutic efficacy, but little is known about the induction, functionality and persistence of tumor-specific memory responses. To our knowledge, only one clinical study has described the induction of tumor-specific T-cell responses after catumaxomab (CD3xEpCAM) therapy, which was mediated via engagement of Fc-gamma receptors (FcγRs) on myeloid cells by its active Fc tail.17 Since antigen loss is described as the major escape mechanism for CD3 bsAb treatment of hematological tumors in the clinic, this suggests that insufficient tumor-specific responses were raised to eradicate antigen-negative tumor cells and prevent relapse. In murine tumor models, contradicting results have been reported. One study showed induction of systemic tumor-specific responses following CD3 bsAb therapy in a syngeneic mouse model overexpressing a human tumor antigen, enabling rejection of a second tumor challenge in the absence of further treatment.18 In contrast, we previously showed that protective memory was not installed after CD3 bsAb treatment in the fully syngeneic B16F10 murine tumor model targeting the endogenous tyrosinase-related protein 1 (TRP1) antigen.19 These data suggest that the immunogenicity of the involved tumor antigen or the degree of spontaneous T-cell infiltration in the TME may determine the outcome. This warrants a thorough investigation on the induction of protective tumor-specific immunity during CD3 bsAb therapy in the context of solid tumors.
In this manuscript, we examine multiple combination strategies to improve the therapeutic efficacy against the primary tumor and concomitantly augment the formation of tumor-specific memory responses that protect against rechallenge. We test combinations of CD3xTRP1 with tumor-localized costimulation, Fc-active tumor-targeted monospecific antibodies and cancer vaccination approaches in the immunologically “cold” B16F10 melanoma model and in the immunologically “hot” MC38 colon carcinoma model. Collectively, our findings advocate for a combination of tumor-specific vaccination with CD3 bsAb therapy to improve therapeutic efficacy against primary tumors and to install long-term protective memory in immunologically “hot” as well as “cold” tumors.
Results
CD3xTRP1 bsAb therapy induces weak and short-lived systemic CD8 T-cell immunity
First, mice harboring immunologically “cold” syngeneic B16F10 tumors, endogenously expressing the tumor antigen TRP1, were treated with the Fc-inert mouse CD3xTRP1 bsAb at days 6 and 9 post-tumor challenge (figure 1A). In concordance with our previous results,19 this early treatment delayed tumor outgrowth in all mice and completely cleared tumors in approximately 40% of the mice (figure 1B and online supplemental S1A). However, no protective immunity was installed after this treatment, as rechallenge of the complete responder mice with a second injection with B16F10 tumor cells at the contralateral flank at day 80 did not result in delayed tumor outgrowth compared with age-matched naïve control mice (figure 1A and C and online supplemental S1A). Analysis of T cells in peripheral blood revealed that CD3 bsAb treatment increased central memory (CD44+/CD62L+) CD8, but not CD4, T-cell frequencies at the expense of their naïve (CD44−/CD62L+) counterparts, suggesting therapy-induced T-cell differentiation (figure 1D). Next, we examined the presence of tumor-specific CD8 T-cell responses in peripheral blood by measuring ex vivo cytokine production after a brief co-culture with B16F10 tumor cells. Tumor necrosis factor alpha (TNFα), but not interferon-gamma (IFNγ) production, by CD8 T cells was specifically observed in the blood of CD3xTRP1-treated mice 12 days after treatment initiation (figure 1E), thereby illustrating induction of systemic CD8 T-cell immunity. The failure to produce IFNγ suggested a low activation status of these tumor-specific T cells20 and these responses waned before day 39 (figure 1E), corroborating the lack of long-term protective memory.
Figure 1. CD3xTRP1 monotherapy induces tumor-specific responses, but does not protect from rechallenge. (A) Treatment schedule for mice inoculated with B16F10 tumors and treated with CD3xTRP1 bsAb. (B-C) Kaplan-Meier survival graphs for primary tumor challenge (B) or rechallenge (C). Numbers indicate surviving mice. Age-matched naïve mice were used as untreated controls for rechallenge. (D) Differentiation of T cells in blood 9 days after the final CD3xTRP1 administration. (E) Tumor-specific responses were measured by determining cytokine production in CD8 T cells from blood taken on days 18 or 39 co-cultured with medium or IFN[gamma] prestimulated B16F10 tumor cells. Data represented as mean+-SEM for n=10-24 (D) or n=2-11 (E). Statistics were calculated using Mantel-Cox log-rank tests (B-C), unpaired two-sided t-tests to compare treatments (D-E), or paired two-sided t-tests to compare different stimulations within the same treatment (E). bsAb, bispecific antibody; IFN[gamma], interferon-gamma; T cm , central memory T cell; T em , effector memory T cell; T n , naïve T cell; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
As we did observe some tumor-specific T-cell responses after CD3xTRP1 monotherapy, we aimed to increase the persistence and quality of these T cells. Previous reports indicated that the combination of CD3 bsAb with 4-1BB costimulation enhanced these traits, resulting in superior antitumor activity.18 To this end, a murine Fc-inert 4-1BBxTRP1 bsAb was coadministered together with CD3xTRP1, leading to targeted costimulation at the tumor site (online supplemental figure S1B), as agonistic 4-1BB monospecific antibodies come with the risk of adverse events.21 While this combination treatment improved antitumor activity against the primary tumor compared with CD3xTRP1 monotherapy, the complete responder mice did not display protection against rechallenge compared with age-matched naïve mice (online supplemental figure S1C-G). Together, these results imply that other combinations should be pursued to induce protective systemic immunity in immunologically “cold” tumors.
Combination with a tumor-opsonizing antibody improves primary survival and activates intratumoral APCs, but fails to install protective memory
Next, we examined the induction of tumor-specific T-cell responses by combining CD3 bsAb with tumor-opsonizing antibodies, reasoning that these may promote antigen uptake and cross-presentation by engaging APCs.22 23 To increase the priming of tumor-specific T cells, we first injected the Fc-inert CD3xTRP1, resulting in the lysis of tumor cells and release of tumor debris, followed by administration of the monospecific Fc-active TA99 antibody targeting the same TRP1 epitope, which could engage FcγRs on APCs (figure 2A). This combination treatment of CD3xTRP1 with TA99 enhanced the survival compared with CD3xTRP1 monotherapy, while TA99 alone had no impact on tumor outgrowth (figure 2B and online supplemental figure S2A-D). Surprisingly, a similar tendency for survival benefit was observed when CD3xTRP1 was combined with an Fc-silenced TA99 antibody, indicating that at least part of this antitumor effect was independent of FcγR engagement. Spectral flow cytometry profiling of the TME revealed no differences in the immune cell frequencies for both combinations of bsAb with TA99 compared with CD3xTRP1 monotherapy (online supplemental figure S3A-B and S4A-B). However, phenotypic analysis demonstrated that tumors treated with a combination of CD3 bsAb with Fc-active TA99 contained higher frequencies of CD86-positive macrophages, conventional dendritic cell 1 (cDC1s) and cDC2s, and higher expression of inducible nitric oxide synthase (iNOS) on macrophages, in addition to minor changes in the activation status of Natural Killer cells (figure 2C–D and online supplemental figure S4C-G). These data pointed to the engagement of FcγRs on innate immune cells by the Fc-active TA99 antibody, but did not explain the therapeutic benefit of Fc-inactive TA99 in combination with bsAb. As previous studies have reported a role for TRP1 in tumor cell proliferation,24 25 in addition to its commonly known role in melanin production, we decided to investigate the potential direct effects of TA99 on tumor cells. Indeed, significant inhibition of B16F10 tumor cell growth was detected in vitro in the presence of monospecific TA99 antibodies, irrespective of their capability to bind FcγRs (online supplemental figure S4H). The effect of the bispecific CD3xTRP1 on tumor growth was much smaller, most likely due to the difference in avidity. These results suggested that monospecific (Fc-silenced) TA99 improved the efficacy of CD3 bsAb treatment through direct impairment of tumor cell growth. Furthermore, the TA99 antibody with an active Fc tail enhanced the engagement of the myeloid cell compartment, which might aid the development of tumor-specific T-cell responses.
Figure 2. Combination of CD3xTRP1 with Fc-active TA99 augments primary survival but does not improve tumor-specific T-cell responses. (A) Treatment schedule for mice inoculated with B16F10 tumor cells and treated with a combination of CD3xTRP1 with TA99. (B) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. (C-D) Expression of phenotypical markers on intratumoral macrophages (C), or cDC1s and cDC2s (D) treated according to the treatment scheme in online supplemental figure S4A . Numbers indicate average expression. Statistical significance is only shown for comparisons against the bsAb treatment group. (E) Tumor-specific responses on days 25 and 39 were measured by determining cytokine production in CD8 T cells from blood co-cultured with medium or IFN[gamma] prestimulated B16F10 tumor cells. (F) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. Data represented as mean on a heatmap for n=4-6 (C-D, two untreated mice were excluded from the analysis based on tumor size<27 mm 3 ), or mean+-SEM for n=3-14 (E). Statistics were calculated using Mantel-Cox log-rank tests (B and F), one-way ANOVA followed by Tukey’s post hoc tests to compare all treatments (C-E), or paired two-sided t-tests to compare different stimulations within the same treatment (E). ANOVA, analysis of variance; bsAb, bispecific antibody; cDC1s, conventional dendritic cells 1; cDC2s, conventional dendritic cells 2; IFN[gamma], interferon-gamma; iNOS, inducible nitric oxide synthase; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
We then investigated tumor-specific T-cell responses in the blood of these mice. Lower levels of TNFα and IFNγ were observed in CD8 T cells at day 25 for either of the combination therapies compared with CD3 bsAb monotherapy and again these responses were not detectable anymore on day 39 (figure 2E). When mice that rejected the primary tumor were rechallenged at day 80, no efficient protection against the second B16F10 tumors was observed, with the exception of a marginal delay in tumor outgrowth for the combination of CD3xTRP1 and (Fc-active) TA99 (figure 2F and online supplemental S2A and C). Altogether, these results demonstrate that a monospecific Fc-active tumor-opsonizing antibody could improve the activity of CD3 bsAb therapy against the primary tumor and enhance the activation status of intratumoral APCs, but fails to install long-term protective memory.
Tumor-non-specific vaccines promote therapy response to primary but not secondary tumors
We recently reported that therapeutic synthetic long peptide (SLP) vaccines, containing tumor-unrelated CD8 T-cell antigens and adjuvants, facilitated the accumulation of intratumoral T cells, promoted a proinflammatory TH1-like TME and empowered the therapeutic activity of CD3 bsAbs in solid tumors.26 We wondered whether this combination strategy would also induce tumor-specific T-cell responses and protection from rechallenge. Since multiple studies have demonstrated the importance of CD4 T-cell help in memory formation of T-cell responses,27 28 a CD4 epitope was included in the SLP vaccine in addition to the CD8 epitope. Tumor-non-specific ovalbumin (OVA) SLP vaccines were administered together with TLR9-ligand CpG (Cytosin-phosphatidyc-Guanin) as an adjuvant, followed by CD3xTRP1 treatment. Administration of CD3xTRP1 bsAb was applied on days 12 and 15 (contrasting earlier experiments where days 6 and 9 were used) to first allow vaccine-induced T-cell infiltration into the tumor (figure 3A).26
Figure 3. Combination of CD3xTRP1 with tumor-non-specific vaccination improves primary survival and tumor-specific responses, but does not install protective memory. (A) Treatment schedule for mice inoculated with B16F10 tumor cells and treated with a combination of CD3xTRP1 and SLP vaccination containing OVA CD8 and/or CD4 epitopes and CpG as adjuvant. (B) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. (C) Frequency of OVA Tm + CD8 T cells in blood on days 25 and 45. (D) Tumor-specific responses were measured by determining cytokine production in CD8 T cells from blood co-cultured with medium or IFN[gamma] prestimulated B16F10 tumor cells on day 25. (E) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. Data represented as mean+-SEM for n=1-14 (C) or n=4-14 (D). Statistics were calculated using Mantel-Cox log-rank tests (B and E), one-way ANOVA followed by Tukey’s post hoc tests comparing all groups (C), or comparing all treatments for CD8 T cells stimulated with B16F10 tumor cells (D), or paired two-sided t-tests to compare different stimulations within the same treatment (D). ANOVA, analysis of variance; bsAb, bispecific antibody; CpG, Cytosin-phosphatidyc-Guanin; IFN[gamma], interferon-gamma; OVA, ovalbumin; SLP, synthetic long peptide; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
In line with our previous results,26 OVA CD8 SLP vaccination enhanced the therapeutic efficacy of CD3xTRP1 (figure 3B and online supplemental S5A). Also, the combination of CD3xTRP1 with OVA CD4 or OVA CD8+CD4 SLP vaccine improved antitumor activity, with the latter combination yielding the highest fraction of surviving mice. At day 25, OVA-specific tetramer-positive (Tm+) CD8 T cells were detected in the blood of all treatment groups that received CD8 SLP vaccination (figure 3C). Interestingly, these tumor-unrelated OVA-specific CD8 T cells waned by day 45 in the vaccinated only group, whereas mice receiving combinations of CD8 SLP vaccination with CD3 bsAb still harbored detectable T-cell frequencies, suggesting that the CD3 bsAb promoted T-cell longevity. In all CD8 SLP vaccinated mice, OVA Tm+ CD8 T cells displayed markers indicative of an effector memory phenotype (CD44+/CD62L−) (online supplemental figure S5B). Importantly, higher B16F10-specific responses of peripheral CD8 T cells were observed at day 25 in all groups receiving combination treatment of vaccination with CD3 bsAb compared with single component controls (figure 3D). These findings implied that the vaccination, as well as the CD3 bsAb component, is essential for strong and systemic induction of tumor-specific CD8 T cells, most likely due to tumor cell kill in the presence of immune-stimulating vaccine adjuvant, as we previously unraveled.26 However, tumor-specific T-cell responses were hardly detectable at day 45 in blood samples (online supplemental figure S5C). Accordingly, no protective immunity was observed when the surviving complete responder mice were rechallenged at day 80 (figure 3E and online supplemental S5A).
We concluded that, despite clear induction of tumor-specific T cells, our previously defined combination strategy of T-cell stimulating tumor-non-specific vaccines with CD3 bsAb therapy failed to install protective memory in the immunologically “cold” B16F10 model.
Combination of CD3 bsAb with tumor-non-specific vaccines installs protective memory in the immunologically “hot” MC38 tumor model
As the B16F10 tumor model is notoriously known for its “cold” TME,29 reflected by the low activity of immunotherapeutic treatments such as checkpoint inhibition, we wondered if protective memory would be induced in a more immunogenic tumor model. We selected the MC38 colon carcinoma model, as this is known for its high mutational load and susceptibility to immunotherapeutic interventions. The MC38 cell line was transfected with the TRP1 tumor antigen (MC38.TRP1) to allow targeting by the CD3xTRP1 bsAb. MC38 tumors indeed displayed a denser spontaneous immune cell infiltrate in vivo than the B16F10 tumors, including higher frequencies of CD8 T cells displaying an activated phenotype (online supplemental figure S6). MC38.TRP1 tumors had a similar immune cell infiltrate as wild-type (WT) MC38 tumors.
BsAb monotherapy and combinations with a vaccine comprizing the tumor-non-specific OVA CD8 SLP or the tumor-specific Rpl18 antigen (ribosomal protein L18) CD8 SLP, a strong endogenous neoantigen in this tumor model,30 were tested in the MC38.TRP1 model (figure 4A). CD3xTRP1 monotherapy only slightly delayed tumor outgrowth, whereas combinations with tumor-non-specific OVA or tumor-specific Rpl18 vaccination controlled nearly all tumors (figure 4B and online supplemental S7A-B),26 demonstrating the sensitivity of MC38.TRP1 tumors to this combination therapy compared with B16F10. Interestingly, all outgrowing tumors from CD3 bsAb monotherapy-treated mice lost TRP1 expression, in contrast to those from untreated mice (figure 4C–D and online supplemental S7C), illustrating selective immune pressure by the CD3 bsAb. Importantly, these results implied that the addition of a vaccine prevented escape through antigen loss and effectively eradicated residual TRP1-negative tumor cells. Strikingly, the combination of CD3xTRP1 with the vaccine adjuvant alone (TLR9 agonist CpG) demonstrated a similar survival benefit compared with the combinations with OVA or Rpl18 SLP (figure 4B and online supplemental S7B), suggesting that peptide antigen was redundant in this model. Of note, the adjuvant CpG was injected at the vaccination site at the tail base, and not intratumorally, but was still able to boost the antitumor activity of the tumor-targeting CD3 bsAb.
Figure 4. In the immunologically “hot” MC38.TRP1 model long-term systemic immunity in installed by CD3 bsAb in combination with a vaccine adjuvant. (A) Treatment schedule for mice inoculated with MC38.TRP1 tumor cells and treated with a combination of CD3xTRP1 and vaccination with tumor-non-specific OVA or tumor-specific Rpl18 CD8 SLP epitopes and CpG as an adjuvant. (B) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. (C-D) TRP1 expression of end-stage MC38.TRP1 tumors are visualized as exemplary histograms (C) and bar graphs (D). (E-F) Analysis of Rpl18 tetramer CD8 T-cell responses and phenotype in the blood on day 25 (E) or day 39/42 (F). Phenotypical data is only shown for mice with>0.2% of Rpl18 Tm + from CD8 T cells (E). The dotted line indicates the mean Rpl18 Tm + frequency + two SD of the naïve mice. (G-H) Tumor-specific responses were measured by determining cytokine production in CD8 T cells from blood co-cultured with medium, or IFN[gamma] prestimulated MC38 or MC38.TRP1 tumor cells on day 25 (G) or day 39/42 (H). (I-J) Kaplan-Meier survival graphs for mice receiving sequential rechallenges with MC38.TRP1 (I) and MC38 tumors (J). (K) Kaplan-Meier survival graphs for mice receiving a rechallenge with MC38 in the presence of CD8-depleting antibodies. Numbers indicate surviving mice. Data represented as mean+-SEM for n=3-7 (D), n=7-23 (E, G), or n=3-24 (F, H). Statistics were calculated using Mantel-Cox log-rank tests (B and I-K), using one-way ANOVA followed by Tukey’s post hoc tests comparing all groups (D-F), comparing all treatments for cells stimulated with MC38.TRP1 tumor cells (G-H), or using repeated measures ANOVA followed by Dunnett’s post hoc tests compared with medium to compare different stimulations within the same treatment (G-H). ANOVA, analysis of variance; bsAb, bispecific antibody; CpG, Cytosin-phosphatidyc-Guanin; FMO, Fluorescence Minus One; IFN[gamma], interferon-gamma; MFI, Mean Fluorescence Intensity; OVA, ovalbumin; Rpl18, ribosomal protein L18; SLP, synthetic long peptide; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
We then interrogated antitumor T-cell responses and first measured Rpl18 Tm+ T-cell frequencies in blood. As expected, Rpl18 Tm+ frequencies were high in mice after Rpl18 vaccination, while the other groups showed very low percentages which tended to be higher than in naïve mice (figure 4E–F). The Rpl18 Tm+ CD8 T cells from all treated mice demonstrated higher expression of proliferation marker ki67 compared with untreated mice (figure 4E and online supplemental S7D-F), suggesting the presence of the MC38.TRP1 tumor already induced low levels of Rpl18-specific CD8 T cells, while treatment promoted the proliferation of these cells. Analysis of immune cells from end-stage tumors confirmed this notion, as clearly detectable frequencies of Rpl18 Tm+ CD8 T cells were present in mice that did not receive Rpl18 vaccination (online supplemental figure S7G). Next, the antitumor activity of CD8 T cells in blood was assessed following co-culture with WT or TRP1-transfected MC38 tumor cells. On day 25, the strongest MC38-directed T-cell responses were found in mice treated with tumor-specific Rpl18 vaccination alone or together with CD3 bsAb (figure 4G and online supplemental S7H). As expected, T-cell responses to WT MC38 tumor cells were much stronger after (combination) treatment with the Rpl18 neoantigen vaccine than for CD3xTRP1 monotherapy or its combination with OVA vaccine, or adjuvant alone (figure 4G and online supplemental S7H). Moreover, CD3xTRP1 treatment stimulated the induction of T-cell responses towards the MC38.TRP1 tumor, which was most pronounced in combination with Rpl18 or OVA SLP vaccine. Finally, except for the Rpl18-vaccinated mice, WT MC38-specific and MC38.TRP1-specific T-cell responses waned around day 40 (figure 4H). These data demonstrated the induction of some Rpl18-specific CD8 T cells following MC38.TRP1 tumor inoculation, a strong and lasting tumor-directed response after Rpl18 vaccination and a waning response for all other treatments.
Finally, we rechallenged mice that rejected the primary tumor with MC38.TRP1 at the opposite flank and observed full protection in nearly all mice (figure 4I and online supplemental S7A). To test whether the protection was directed towards the transfected TRP1 antigen or endogenous MC38 antigens, we rechallenged the surviving mice again, but now with WT MC38 tumor cells. Again, almost all mice fended off this tumor rechallenge (figure 4J and online supplemental S7A), illustrating the successful induction of protective immunity towards endogenous MC38 antigens. Importantly, protection from tumor rechallenge was completely ablated on CD8 T-cell depletion, delineating the role of tumor-specific CD8 T-cell responses in protective memory (figure 4K and online supplemental S7I).
Together, these findings show that T-cell inducing vaccines, and even their adjuvants, improve the efficacy of CD3 bsAb therapy and simultaneously induce protective tumor-specific immune responses in the “hot” MC38 tumor model.
Tumor-specific vaccines with CD3 bsAb install protective memory in the “cold” B16F10 model
We next examined whether the combination of a tumor-specific vaccine with CD3xTRP1 antibody would result in protective memory in a “cold” tumor model. For that, we used B16F10 cells expressing OVA as a tumor-specific non-self antigen. No significant differences in the frequency or phenotype of intratumoral CD8 T cells were observed between untreated B16F10 and B16F10.OVA tumors, despite the introduction of the immunogenic OVA antigen (online supplemental figure S8A). To ensure high frequencies of tumor-specific T cells, we first included adoptively transferred transgenic OT-1 CD8 T cells (recognizing the OVA antigen), followed by vaccination with OVA CD8 SLP and CD3xTRP1 (online supplemental figure S8B). All mice receiving this triple combination therapy cleared the primary tumors (online supplemental figure S8C) and retained high frequencies of tumor-specific OT-1 T cells in the blood at day 79, indicative of long-term survival of these cells (online supplemental figure S8D). Importantly, all mice in the triple combination group were also protected from rechallenge with B16F10.OVA cells, whereas approximately 70% of the mice receiving OT-1 transfer and vaccination were protected (online supplemental figure S8E). This demonstrated that protective memory can be induced in the context of CD3 bsAb treatment in this melanoma model.
Next, OT-1 T-cell transfer was omitted from the treatment regimen (figure 5A) and the antitumor activity of the combination treatment of CD3 bsAb with OVA CD8 and/or CD4 SLP vaccination was tested in the B16F10.OVA tumor model. The combination of CD3 bsAb with OVA CD8+CD4 SLP vaccination displayed the strongest survival benefit, while the individual OVA CD8 and CD4 SLP vaccination also improved CD3 bsAb therapeutic efficacy (figure 5B and online supplemental figure S8F). Monotherapy vaccination with OVA CD8+CD4 SLP (but without CD3 bsAb) also delayed tumor growth, supporting the notion that OVA is a strong non-self tumor antigen. Analysis of blood samples on day 24 and day 39 revealed that the groups receiving OVA CD8 vaccination showed strong and lasting induction of OVA-specific Tm+ CD8 T cells with predominantly an effector memory phenotype (figure 5C and online supplemental figure S8G). When comparing the OVA Tm+ CD8 T cells in blood between the different experiments containing tumor-non-specific (B16F10) and tumor-specific (B16F10.OVA) vaccination, we found no differences at the early time point (day 24/25), suggesting that these responses were mainly vaccine-mediated (online supplemental figure S8G). However, at the late time point (day 39/45), mice bearing B16F10.OVA tumors displayed higher frequencies of OVA Tm+ CD8 T cells with a central memory phenotype (online supplemental figure S8G). Analysis of cytokine production by peripheral blood T cells co-cultured with tumor cells revealed strong and durable CD8 T-cell responses towards B16F10.OVA after OVA CD8 SLP vaccination, whereas responses towards WT B16F10 tumor cells were less pronounced and lost over time, in line with our previous results (figure 5D–E). Importantly, the combination of CD4+CD8 SLP vaccination with CD3 bsAb treatment induced long-term protection to rechallenge with B16F10.OVA tumor cells on day 80 (figure 5F and online supplemental S8F). Of note, the two complete responder mice after the OVA CD8 SLP+bsAb treatment both rejected the rechallenge as well, in contrast to the two complete responder mice in the CD4 SLP+bsAb group, speculating that the CD8 T cells are essential for protection against rechallenge (online supplemental figure S8F). When the mice that completely rejected the first rechallenge with B16F10.OVA was again rechallenged with WT B16F10 tumor cells, no protection was observed, indicating that despite the clearance of two OVA-positive tumors, no protective immune responses were induced against endogenous antigens (figure 5F and online supplemental figure S8F).
Figure 5. In the B16F10.OVA tumor model, combination of CD3xTRP1 with a tumor-specific vaccination improves primary survival and installs protective memory responses. (A) Treatment schedule for mice inoculated with B16F10.OVA tumor cells and treated with a combination of CD3xTRP1 and SLP vaccination with OVA CD8 and CD4 epitopes. (B) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. (C) Frequency of OVA Tm + CD8 T cells in blood on day 24. (D-E) Tumor-specific responses were measured by determining cytokine production in CD8 T cells from blood co-cultured with medium, or IFN[gamma] prestimulated B16F10 or B16F10.OVA tumor cells on day 24 (D) or day 39 (E). (F) Individual tumor outgrowth curves and Kaplan-Meier survival graphs for tumor rechallenges of indicated groups. Arrows indicate tumor cell inoculations, numbers indicate surviving mice. Data represented as mean+-SEM for n=6-14 (C-D), or n=1-13 (E). Statistics were calculated using Mantel-Cox log-rank tests (B, F), one-way ANOVA followed by Tukey’s post hoc tests comparing all groups (C), or comparing all treatments for cells stimulated with B16F10.OVA tumor cells (D-E), or repeated measures ANOVA followed by Dunnett’s post hoc tests compared with medium to compare different stimulations within the same treatment (D-E). ANOVA, analysis of variance; bsAb, bispecific antibody; IFN[gamma], interferon-gamma; n.s., not significant; OVA, ovalbumin; SLP, synthetic long peptide; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
Then, we corroborated the finding that tumor-specific vaccination before CD3 bsAb therapy installs protective immunity by exploiting the endogenous tumor-associated Gp100 antigen instead of the immunogenic non-self OVA antigen in the WT B16F10 tumor model. We used a previously established protocol in our lab using a Gp100 CD8 SLP vaccine, adjuvanted with TLR7/8 agonist imiquimod and interleukin (IL)-2 (figure 6A).26 31 As expected, the addition of Gp100 vaccination to CD3xTRP1 therapy improved survival percentages (figure 6B and online supplemental figure S8H). This combination treatment also increased the frequency of cytokine-producing CD8 T cells in the blood after brief co-culture with B16F10 tumor cells on day 25, which was low but still detectable on day 39 (figure 6C). Importantly, rechallenging the complete responder mice with B16F10 tumor cells resulted in significantly delayed tumor outgrowth when compared with naïve control mice (figure 6D and online supplemental figure S8H).
Figure 6. Combination of CD3xTRP1 with tumor-specific vaccination improves survival and installs protective memory responses against B16F10 tumors. (A) Treatment schedule for mice inoculated with B16F10 tumor cells and treated with a combination of CD3xTRP1 and SLP vaccination with a Gp100 CD8 epitope. (B) Kaplan-Meier survival graphs for indicated groups, numbers indicate surviving mice. (C) Tumor-specific responses were measured by determining cytokine production in CD8 T cells from blood co-cultured with medium, or IFN[gamma] prestimulated B16F10 tumor cells on days 25 or 39. (D) Kaplan-Meier survival graphs for rechallenge of indicated groups, numbers indicate surviving mice. (E) Treatment schedule for mice inoculated with B16F10 tumor cells and treated with a combination of CD3xTRP1, adoptive cell transfer of Pmel T cells and SLP vaccination with a Gp100 CD8 epitope. (F) Individual tumor outgrowth curves for indicated groups, numbers indicate surviving mice. (G) Frequencies of CD45.1 + Pmel T cells in the blood on day 17. (H) Kaplan-Meier survival graphs for rechallenge of indicated groups, numbers indicate surviving mice. Data represented as mean+-SEM for n=2-15 (C), or n=4-8 (G). Statistics were calculated using Mantel-Cox log-rank tests (B, D and H), paired two-sided t-tests to compare different stimulations within the same treatment (C), or one-way ANOVA followed by Tukey’s post hoc tests comparing all treatments for cells stimulated with B16F10 tumor cells (C), or comparing all groups (G). ANOVA, analysis of variance; bsAb, bispecific antibody; IFN[gamma], interferon-gamma; n.s., not significant; SLP, synthetic long peptide; TNF[alpha], tumor necrosis factor alpha; TRP1, tyrosinase-related protein 1.
To assess whether this protective memory could be further improved by increasing the frequency of tumor-specific T cells, we adoptively transferred T cell Receptor-transgenic Gp100-specific CD8 T cells (Pmel) in addition to the treatment regimen (figure 6E). This triple combination therapy was very effective against primary B16F10 tumors, as six out of eight mice showed a complete response (figure 6F). High frequencies of tumor-specific Pmel T cells were detected in the blood of mice receiving the triple treatment (figure 6G). However, rechallenging the complete responder mice did not result in complete protection but only in delayed tumor outgrowth for the majority of the mice (figure 6H), indicating that additional factors are at play in this immunologically “cold” tumor model.
Altogether, these findings show that CD3 bsAb therapy benefits from combination with cancer vaccines to improve therapeutic efficacy and, simultaneously, install protective memory. For immunologically “hot” tumors, combinations with innate activators like TLR agonists alone might be sufficient, whereas “cold” tumors seem to depend on T-cell inducing cancer-specific vaccines.
Discussion
Despite the fact that tumor recurrences are a challenge for the successful treatment of hematological malignancies by CD3 bsAbs, very few publications investigate the emergence of tumor-specific T-cell responses as a result of the therapy, which are capable of mediating long-term tumor protection. We investigated the formation of immunological memory during CD3 bsAb therapy, testing several combination strategies in two mouse tumor models. We show that combination with agonistic costimulatory antibodies, tumor-opsonizing antibodies and tumor-non-specific T-cell stimulating vaccines all improved the therapeutic efficacy of CD3 bsAb treatment when treating primary immunologically “cold” tumors, but did not concomitantly install protective memory against rechallenges. Although TLR agonists in combination with CD3 bsAb were sufficient in immunologically “hot” tumors to reach long-term systemic immunity, the combination of CD3 bsAb with T-cell stimulating tumor-specific vaccines was necessary to prevent recurrence in “cold” tumors.
Two previous reports discussed the induction of protective immune responses in the context of Fc-inert CD3 bsAb therapy. In the first, we demonstrated a lack of protective memory following CD3 bsAb treatment using the immunologically “cold” B16F10 tumor model,19 whereas the second publication showed successful protection from rechallenge using the TRAMP-C2 tumor model, transfected with the xenogeneic human prostate-specific membrane antigen (PSMA).18 In contrast to B16F10, TRAMP-C2 tumors were shown to display high levels of T-cell infiltrate. This difference is also observed in our current study, where CD3 bsAb monotherapy induced protection from rechallenge in the well-infiltrated “hot” MC38.TRP1 tumor (online supplemental figure S7A), but not in B16F10 (figure 1C). We speculate that the heterologous expression of the human PSMA in the TRAMP-C2 model might contribute to its immunogenicity and degree of T-cell infiltration.
Two additional publications reported Fc-mediated induction of broad antitumor immunity in patients receiving CD3 bsAbs comprizing an active Fc backbone, enabling FcγR and complement binding.17 22 These antibodies can engage FcγR on APCs to promote antigen spread, similar to the above-reported experiments in which we coadministered an Fc-active monospecific TA99 antibody. However, these Fc-active CD3 bsAbs have a high toxicity profile due to Fc-mediated crosslinking of T cells independent of the tumor antigen.32 Therefore, T-cell engagers are now generally developed without an Fc tail or with an Fc-inert backbone. Further research is warranted to achieve a similar induction of tumor-specific responses on combining an Fc-inert CD3 bsAb and monospecific Fc-active Ab as was observed for the Fc-active bsAb. A combination of a CD3 bsAb (CD3xBCMA) with an Fc-active monospecific antibody (CD38) is currently studied in a phase 3 clinical trial in multiple myeloma (NCT05083169) after promising results in previous studies.33–35 Additionally, multiple phase 1/2 studies in patients suffering from B-cell malignancies investigate combinations of CD19-targeting and CD20-targeting CD3 bsAbs with standard-of-care R-CHOP treatment, which includes rituximab (CD20) monospecific antibody (NCT03931642, NCT05798156, NCT05800366, NCT04623541).
Here, we show that CD3 bsAb therapy can best be combined with tumor-specific T-cell stimulating vaccines to improve antitumor activity against the primary tumor and simultaneously induce protective memory responses against immunologically “cold” as well as “hot” tumors. It has been suggested that CD3 bsAb therapy results in strong activation of intratumoral T cells, leading to activation-induced cell death, T-cell dysfunction or T-cell exhaustion.36–38 We hypothesize that immunogenic tumors recruit more T cells due to a better endogenous antitumor immune response and might thereby provide a continued influx of functional T cells from the periphery and that these cells mediate rapid eradication of tumor cells in the recurrence phase. This could also be achieved for tumors with a “cold” immunologic environment by administration of tumor-specific vaccines, which generate a source of fresh T cells that traffic to tumors.26 These T cells can persist long-term in the blood and lymph organs as a pool of tumor-specific memory T cells, responsible for immediate rejection of tumor cells at rechallenge.
Nearly all treatments described in this manuscript induced some level of tumor-specific T-cell responses, but their degree depended on the (combination) treatment. However, the presence of tumor-specific T-cell responses in blood did not seem to be fully predictive for protection against rechallenge. Despite low levels of circulating tumor-specific T cells at later time points, most complete responder mice were protected from rechallenge in the MC38 model. Interestingly, Rpl18 Tm+ T cells were clearly present in the tumors that grew out under treatment, suggesting that T-cell numbers in blood might not reflect the memory capacity induced by the therapy. The location of our rechallenge differed from that of the primary tumors (opposite flank), excluding the involvement of tissue-resident memory T cells, a dedicated subset of memory cells retained in the local tissue of infliction.39 We assume that circulating systemic tumor-specific memory T cells are reactivated at the time of rechallenge, which was sufficient for fast clearance of the second tumor.
The survival studies in the MC38.TRP1 tumor model revealed that all tumors treated with CD3 bsAb escaped therapy control via loss of TRP1 expression. Most likely, this was not active loss of the transfected gene, but merely outgrowth of the low frequency of TRP1-negative cells present in the injected tumor cells. Nonetheless, this strong selective pressure mediated by CD3 bsAb therapy is highly reminiscent of the clinical phenomenon of therapy resistance.11–16 Strikingly, combination therapy of CD3 bsAb with vaccination prevented the outgrowth of antigen-negative tumor variants and successfully treated nearly all primary tumors, implying that this combination therapy is able to mediate the eradication of TRP1-negative MC38 tumor cells. The small fraction of WT MC38 tumor cells could be eradicated by Rpl18-specific T cells, which are primed by tumor inoculation and further boosted by vaccine adjuvant and CD3 bsAb therapy (figure 4E–F).30 In our previous study focused on the mechanism of action of T-cell stimulating vaccines with CD3 bsAb therapy, we revealed a strong T-cell influx in tumors, which were subsequently activated by the bsAb, leading to enhanced T-cell activation and a Th1-skewed TME landscape.26 After combination therapy, activation of innate immune cells, including perforin-positive NK cells and iNOS-positive macrophages, was observed. Moreover, we showed that this combination therapy depended on the T-cell induced imprinting of the local macrophages.40 The involvement of innate immune cells to eradicate antigen-negative MC38 tumor cells would fit with recent work of us and others, where innate cells such as macrophages, neutrophils and eosinophils were crucial for therapeutic responses following T-cell based immunotherapies.40–42 Interestingly, we showed that the protection from tumor rechallenge was mediated by CD8 T cells, as depletion of these immune cells abrogated long-term memory, indicating that the therapy-induced acute rejection of tumors is a coordinated attack involving innate and adaptive responses, while the memory response resides in CD8 T cells.
We advocate for the clinical investigation of using tumor-specific T-cell stimulating cancer vaccines before CD3 bsAb therapy due to the superior efficacy against existing tumors and the installation of systemic immunological memory, thereby preventing recurrences. In general, cancer vaccines have delivered disappointing results and failed to eradicate established diseases, although some were shown to be effective in a premalignant or minimal residual disease stage.43–45 However, recent developments are promising, as personalized messenger RNA vaccines targeting multiple neoantigens have demonstrated therapeutic benefits against immunologically “cold” cancers.46 47 These vaccines were developed in a patient-specific manner by exome sequencing the tumors, thereby illustrating broad applicability and individual tailoring. The survival benefit of such vaccines could potentially be even further enhanced on combination with other immunotherapies, such as CD3 bsAbs or immune checkpoint blockade, of which the latter combination is currently investigated in a phase 3 clinical trial in patients with melanoma (NCT05933577).
To conclude, we have shown that the combination of CD3 bsAb with tumor-specific vaccination strongly improved survival against the primary tumor and generated durable vaccine-induced tumor-specific responses that could protect from rechallenge. Further research into such combination therapies might help to improve the efficacy of CD3 bsAb therapy in solid cancers and prevent metastases and recurrences in patients with cancer.
Inclusion and diversity
We support inclusive, diverse, and equitable conduct of research.
Methods
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, TvH, ([email protected]).
Materials availability
This study did not generate unique new reagents, all materials are listed in online supplemental file 1.
Data and code availability
Any additional information required to reanalyze the data reported in this paper is available from the lead contact on request.
Experimental model details
Mice
Eight weeks old C57BL/6 mice were purchased from Charles River, the Netherlands. Transgenic mice containing a TCR specific for chicken ovalbumine OVA257–264/H-2Kb (designated as OT-1) were purchased from Jackson Laboratories (stock number 003831) and bred in house to express the congenic CD45.1 marker. Transgenic mice containing a TCR specific for Gp10025-33/H-2Db (designated as Pmel) were kindly provided by Dr N P Restifo (Center for Cancer Research, National Cancer Institute, National Institute for Health, Bethesda, Maryland, USA) and bred to express the congenic CD45.1 marker. All mouse experiments were performed using∼8 weeks old male mice at the animal facility of the Leiden University Medical Center (LUMC), the Netherlands after∼1 week of acclimatization. Sample sizes for survival studies were calculated using power and sample size software, using a power of 0.80, a type I error probability of 0.05 and the expected difference in survival between the treatment groups based on previous experiments. Sample sizes for TME studies were based on experience with the model. All outcome measures are shown in the (supplementary) figures. Mice were distributed by hand over the various treatment groups based on tumor volume, or randomly prior to the start of the experiment if tumors would be too small to distribute based on tumor volume at the moment of treatment. In both cases multiple treatment groups were present in each cage to account for cage effects and introduce some form of blinding, as the researchers were mostly aware of the treatments in each cage. The health status of the animals was monitored over time and all animals tested negative for agents listed in the FELASA (Federation of European Laboratory Animal Science Associations) guidelines for specific-pathogen free mouse colonies.48 All mouse studies were approved by the Dutch animal ethics committee (CCD, Centrale Commissie Dierproeven) and the local Animal Welfare Body of the LUMC on the permit numbers AVD116002015271 and AVD11600202010004. Experiments were performed in accordance with the Dutch Act on Animal Experimentation and EU Directive 2010/63/EU (“On the protection of animals used for scientific purposes”).
Cell lines
The B16F10 mouse melanoma cell line was purchased from the American Type Culture Collection (CRL-6475). B16F10.OVA expressing full-length OVA protein49 and the chemically induced murine colon carcinoma cell line MC38 (Kerafast)50 were kindly provided by Prof F Ossendorp (LUMC, Leiden, the Netherlands). MC38.TRP1 was generated as previously described51 and was cell-sorted for TRP1 surface expression using the anti-TRP1 antibody TA99. Unless indicated otherwise, all cell lines were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen) supplemented with 8% Fetal Calf Serum (Greiner), 2% penicillin/streptomycin (Gibco) and 2 mM glutamine (Gibco) at 37°C and 5% CO2. MC38 cell lines and B16F10.OVA was further supplemented with 1× non-essential amino acids (Gibco) and 1 mM sodium pyruvate (Life Technologies), with MC38.TRP1 additionally received 400 µg/mL neomycin (G418, Life Technologies) and B16F10.OVA additionally received 1 mg/mL neomycin and 60 µg/mL hygromycin (Life Technologies). Cell lines were authenticated by IDEXX BioAnalytics using short tandem repeat markers, frequently tested for mycoplasma and tested for known mouse pathogens using Mouse Antibody Production tests for in vivo use.
Method details
Antibody, peptide and tetramer generation
Monospecific TA99 and Fc-silenced TA99 were generated in-house by Genmab by transfection of 293 F cells with expression vectors containing the relevant heavy and light chains.52 LALAPG mutations were introduced to the Fc domain to generate Fc-silenced TA99.53 CD3xTRP1, 4-1BBxTRP1 and B12xCD3 bsAbs were generated in-house by Genmab with controlled Fab-arm exchange using the 145-2 C11 clone recognizing CD3ε, the TA99 clone recognizing TRP1, the 3H3 clone recognizing 4-1BB and the B12 clone recognizing an HIV epitope.52 54 BsAbs were generated as IgG2a isotypes with L234A-L235A (LALA) mutations to silence Fc-mediated effector functions.55
Peptides for tetramers and immunization were produced in-house using solid-phase peptide synthesis.56 APC-conjugated H-2Kb OVA or Rpl18 tetramers were generated as previously described using SIINFEKL or KILTFDRL peptides, respectively.57
Mouse treatments for survival and infiltration studies
For survival studies involving early CD3xTRP1 administration (days 6 and 9 in the absence of vaccination strategies), mice were injected subcutaneously (s.c.) in the right flank with 80,000 B16F10 tumor cells. For survival studies involving late CD3xTRP1 administration (day 12 and 15 or later in the presence of vaccination strategies), mice were injected s.c. in the right flank with 50,000 B16F10 or 80,000 B16F10.OVA tumor cells. For survival studies involving MC38 or MC38.TRP1 tumor cells, mice were injected with 500,000 tumor cells s.c. in the right flank. Rechallenges were performed by injecting tumor-free mice surviving the initial tumor challenge with 40,000 B16F10, 80,000 B16F10.OVA, or 400,000 MC38 or MC38.TRP1 tumor cells s.c. in the contralateral flank to prevent tumor rejection from tissue-resident T cells and always contained a naïve age-matched control group. For TME studies, mice were injected with 150,000 B16F10 or B16F10.OVA, or 500,000 MC38 or MC38.TRP1 tumor cells s.c. in the right flank. All tumor cells were injected in 200 µL phosphate-buffered saline (PBS) (Fresenius Kabi) containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich). Tumor sizes were measured three times a week by caliper and calculated by multiplying length × height × width. Mice were euthanized when one of the humane endpoints was reached: Tumors exceeded 1,000 mm3, ulcerated tumor, or body conditioning score of 2 or lower. In the case mice received a third tumor challenge, a maximum tumor volume of 200 mm3 was used.
For antibody treatments, mice were injected with 12.5 µg CD3xTRP1, 12.5 µg (online supplemental figure S1C-D) or 50 µg (online supplemental figure S1E-G) 4-1BBxTRP1, 200 µg TA99, or 200 µg Fc-silenced TA99 intraperitoneally (i.p.) in 100 µL PBS.
For vaccination approaches, all peptides for immunizations were produced in-house and performed with CpG as an adjuvant unless indicated otherwise. Mice were injected s.c. in the tail base with 100 µg chicken ovalbumine OVA241-270 peptide (“OVA” or “OVA CD8”) (SMLVLLPDEVSGLEQLESIINFEKLTEWTS), OVA323-341 peptide (“OVA CD4”) (ISQAVHAAHAEINEAGRK), or Rpl18115–132 neoantigen peptide (KAGGKILTFDRLALESPK),30 each supplemented with 20 µg CpG in 50 µL PBS. When mice received both OVA CD8 and CD4, 100 µg of each peptide was included.
Immunizations with Gp100 peptide alone, or in combination with Pmel T cell transfer (figure 5 and online supplemental figure S7), or OVA CD8 peptide in combination with OT-1 T cell transfer (online supplemental figure S6A-B) were supplemented with imiquimod as an adjuvant and IL-2. For these immunizations, mice were anesthetized by an i.p. injection of a mixture of ketamine (60 mg/kg) and medetomidine (0.4 mg/kg), followed by shaving of the flanks and injection of 150 µg Gp10020–39 peptide (AVGALKVPRNQDWLGVPRQL homologous human sequence) or OVA CD8 peptide in 100 µL PBS in the left flank. Then, 60 mg of 5% imiquimod-containing cream Aldara (3M Pharmaceuticals) was simultaneously topically applied on the skin at the injection site and the mice were left for 2–3 hours on heating pads to recover and allowed the cream to be absorbed into the skin. Next, the anesthesia was antagonized by injecting the mice with 1 mg/kg atipamezole in 100 µL PBS i.p. Recombinant human IL-2 (600,000 IU, Proleukin, Clinigen) was injected i.p. in 100 µL PBS on the day of the second immunization and 1 day later.
For T-cell transfer, lymphocytes of spleens and lymph nodes of naïve TCR transgenic OT-1 or Pmel T cells were isolated and enriched for T lymphocytes by nylon wool. 1×106 enriched splenocytes were injected intravenously into the tail vein in 200 µL PBS.
Flow cytometry
Tumors or blood were harvested and immune cells were analyzed with flow cytometry. Single-cell suspensions from MC38 or MC38.TRP1 tumors were prepared by cutting the tumors in small fragments followed by 10 min of incubation with 2.5 mg/mL Liberase (Roche) at 37°C in a humidified atmosphere containing 5% CO2. Then, cell suspensions from MC38 and MC38.TRP1 tumors, or whole B16F10 or B16F10.OVA tumors and spleens were mashed through a 70 µm cell strainer (Falcon) and plated for flow cytometry staining. For blood samples, red blood cells were lysed using lysis buffer (in-house pharmacy) prior to FACS staining. Mouse Fc-receptors were blocked by Rat Anti-Mouse CD16/CD32 (Clone 2.4G2, BD) for 15 min at 4°C. Viability was assessed with the Zombie UV Fixable Viability Kit (BioLegend) or the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit in PBS before surface staining. APC-conjugated H-2Kb OVA257–264 SIINFEKL, or H-2Kb Rpl18119–126 KILTFDRL were stained for 30 min at room temperature (RT) in PBS supplemented with 0.5% BSA+0.002% sodium azide (in-house pharmacy) (FACS buffer). Then, other surface markers were stained in FACS buffer for 20 min at 4°C. Next, cells were fixed and permeabilized for intracellular marker staining using the FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher) according to the manufacturer’s protocol. Finally, cells were resuspended in FACS buffer and measured on a Fortessa cytometer (BD Bioscience), or Aurora 5 L spectral flow cytometer (Cytek) and analyzed using FlowJo software V.10.8.1 (Tree star), or OMIQ software, respectively. An overview of all the antibodies used for flow cytometry is shown in the resources table.
Measurement of tumor-specific T-cell responses in blood
For the measurement of tumor-specific T-cell responses, blood samples were drawn from mice at the indicated time points, followed by lysis of red blood cells using lysis buffer. Subsequently the samples were divided for direct analysis of frequency and phenotype of tumor-specific T cells by flow cytometry as described above, or for co-culture with tumor cells to assess their functionality. For the latter, the lysed blood was co-cultured with 30 000 irradiated tumor cells (6000 Rad) that were prestimulated for 2 days with 30 IU/mL IFNγ (BD Biosciences) to upregulate antigen presentation in the presence of 1 µg/mL GolgiPlug (BD Bioscience) in a 96 w round bottom plate and incubated overnight at 37°C and 5% CO2. The next morning, samples were stained for surface markers and intracellular IFNγ and TNFα as described above.
Analysis of TRP1 expression and Rpl18 Tm in end-stage MC38.TRP1 tumors
Tumors were extracted from mice in survival studies that have reached their humane endpoints during the primary tumor setting. These tumors were cut, enzymatically processed and minced through a cell strainer as described above and then frozen in freezing buffer (50% FCS, 40% IMDM and 10% Dimethylsulfoxide) and stored in liquid nitrogen until use. Then, the samples were thawed and stained for flow cytometry as described above. For the analysis of TRP1 expression, antibodies were labeled with TA99 antibody, followed by a goat-anti-mouse detection step. To correct for the presence of other murine antibodies in the tumor, we also performed staining without TA99 and subtracted this from the full staining.
Crystal violet staining
B16F10 tumor cells (2,900) were plated in 1 mL medium in the presence of 0.1, 1 or 10 µg/mL Fc-active TA99, Fc-silenced TA99, CD3xTRP1 or IgG2a isotype control (B12xCD3) in flat bottom 48 w plates and incubated for 4 days at 37°C and 5% CO2. Then, the medium was removed and cells were fixed with 200 µL ice-cold methanol (Merck) for 10 min at −20°C. Subsequently, methanol was removed and cells were incubated with 200 µL 0.5% crystal violet solution (0.5 g crystal violet powder (Sigma-Aldrich), 80 mL H2O (in house pharmacy) and 20 mL methanol) for 20 min at RT. Next, the crystal violet solution was removed and plates were washed 4× with tap water. Then, plates were dried for at least 30 min. Finally, cell-bound crystal violet was dissolved by adding 100 µL methanol to the well and shaking the plates for 20 min at RT on a bench rocker and quantified by measuring the optical density at 570 nm using a plate reader.
Immunofluorescence microscopy
Tumors were isolated from mice, cut into slices with razor blades and directly fixed in formalin, followed by embedding in paraffin. Tumor tissues were sliced into 4 µm sections and mounted on adhesive slides (VWR). Sections were then deparaffinized and rehydrated, after which endogenous peroxidase activity was blocked using 0.3% hydrogen peroxidase solution (Merck) in methanol (Merck) for 20 min. Antigen retrieval was performed in 0.01 M sodium citrate solution (pH=6.0, Merck) in the microwave for 10 min. Non-specific binding of the primary antibody was blocked by SuperBlock (PBS) Blocking Buffer (Thermo Fisher) at room temperature for 30 min. Tumor slides were then incubated with rat anti-mouse CD8 (clone 4SM15, eBioscience) and rabbit polyclonal CD45 (cat. #ab0558, Abcam) antibodies diluted in PBS at room temperature overnight. Slides were then washed (PBS/0.05% Tween) and incubated with donkey anti-rabbit IgG (H+L) Alexa Fluor 555 (Invitrogen) or donkey anti-rat IgG (H+L) Alexa Fluor 488 (Invitrogen) highly cross-absorbed secondary antibody diluted in PBS at room temperature for 60 min. Nuclear counterstaining was performed with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher) at room temperature for 15 min in the dark. Immunofluorescence images were acquired using the Vectra 3.0.5 multispectral imaging microscope (Perkin Elmer) at 20× magnification. InForm analysis software V.2.6 (Perkin Elmer) was used for the spectral unmixing. Quantification was performed in QuPath V.0.4.3. The total amount of cells was determined using cell detection, after which the amount of CD45+ cells was obtained using the object classifier and the amount of CD8+ cells was calculated using manual counting.
Quantification and statistical analysis
Statistical tests and group sizes (n) are described in the figure legend and calculated between two groups using an unpaired two-sided student’s t-test and between more than two groups using an analysis of variance with Tukey’s post hoc test, unless otherwise indicated. For TME analyses, mice with tumors<27 mm3 were excluded. Unless otherwise indicated in the figure legends, there were no exclusions from statistical analyses. Survival analyses were performed using a log-rank Mantel-Cox test. GraphPad Prism (V.10.1.2) was used for all statistical testing. Data are represented as mean±SEM unless indicated otherwise. Statistical significance is shown as *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
We thank Anouk Stolk and Camilla Labrie for practical help and input regarding the immunofluorescence analyses. Also, we thank Charlotte Berendsen, Amrita Singh and Renoud Marijnissen for project management and Lars Hallander Hansen for intellectual property support. Furthermore, we thank our colleagues from the animal facility, peptide facility and flow cytometry facility in the Leiden University Medical Center. Graphics were created with BioRender.com.
Data availability statement
Data are available upon reasonable request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
Ethics statements
Patient consent for publication
Not applicable.
Ethics approval
Not applicable.
KK and TvH contributed equally.
Contributors Conceptualization: JM, KL, VO, KK and TvH; Methodology: JM and TvH; Formal Analysis: JM, GS and MS; Investigation: JM, GS and MS; Resources: KL, VO, KK and TvH; Writing—Original Draft: JM and TvH; Writing—Review and Editing: JM, KL, VO, SHvdB, KK and TvH; Visualization: JM and TvH; Supervision: KL, VO, JS, SHvdB, KK and TvH; Funding Acquisition: JS, KK and TvH. Guarantor: TvH.
Funding This research was funded by Genmab via a commercial research grant (to TvH).
Competing interests KL, VO, JS and KK are employees of Genmab and have ownership interests (including stock, patents, warrants, etc). JM, KL, VO, JS, KK and TvH are inventors on a patent (WO 2022/049220 A2 entitled Antibody Therapy) involving CD3 bispecific antibody therapy in combination with vaccination. The other authors declare that they have no competing interests.
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.
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Abstract
Background
CD3 bispecific antibody (CD3 bsAb) therapy has become an established treatment modality for some cancer types and exploits endogenous T cells irrespective of their specificity. However, durable clinical responses are hampered by immune escape through loss of tumor target antigen expression. Induction of long-lasting tumor-specific immunity might therefore improve therapeutic efficacy, but has not been studied in detail yet for CD3 bsAbs. Here, we examined multiple combination strategies aiming to improve survival rates in solid tumors and, simultaneously, install endogenous immunity capable of protection to tumor rechallenge.
Methods
Two syngeneic mouse tumor models were employed: The immunologically “cold” B16F10 melanoma and the immunologically “hot” MC38.TRP1 colon carcinoma model. Mice were treated with CD3xTRP1 bsAb (murine Fc-inert immunoglobulin G2a) as monotherapy, or in combination with agonistic costimulatory antibodies, Fc-active tumor-opsonizing antibodies, or tumor-(non)specific vaccines. Treatment efficacy of primary tumors and protection from rechallenge was monitored, as well as induction of tumor-specific T-cell responses.
Results
In the immunologically “cold” B16F10 model, all combination therapies improved antitumor activity compared with CD3 bsAb monotherapy and induced systemic tumor-specific T-cell responses. However, this endogenous T-cell immunity swiftly waned and failed to protect mice from subsequent tumor rechallenge, except for combination therapy with tumor-specific vaccination. These vaccines strongly improved the therapeutic efficacy of CD3 bsAb against primary tumors and led to long-term immunological protection. In the immunologically “hot” MC38.TRP1 model, CD3 bsAb combined with only the vaccine adjuvant was sufficient to generate protective T-cell immunity and, moreover, prevented tumor escape via antigen loss.
Conclusions
These results demonstrate the impact of tumor antigenicity on the induction of protective endogenous antitumor immunity during CD3 bsAb treatment and, importantly, show that the combination with tumor-specific vaccines improves therapeutic efficacy and installs long-term immunological memory in both “hot” and “cold” tumors.
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Details


1 Medical Oncology, Oncode Institute, Leiden University Medical Center, Leiden, The Netherlands
2 Genmab BV, Utrecht, Utrecht, The Netherlands