Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model

Immunotherapy shows promise in treating hematological malignancies by harnessing the immune system's ability to target and kill cancer cells selectively. Unlike traditional chemotherapy and radiation therapy, which damage cells unspecifically, immunotherapies offer more targeted approaches. Various types of immunotherapies, such as monoclonal antibodies (mAb),¹ antibody-drug conjugates (ADCs),² bispecific T-cell engagers (BiTEs),³ immune checkpoint inhibitors,⁴ and chimeric antigen receptor (CAR) T cells (CART),⁵ are currently used in clinical settings. However, response rates and side effects vary among patients,⁶ necessitating further preclinical research to enhance their effectiveness and safety.

Animal models, particularly mouse models, are useful for studying the mechanisms of action and safety of immunotherapies.^7,8 Humanized mice with a functional human immune system provide a physiologically relevant environment for testing human-specific immunotherapies.^9,10 For example, humanized mice have been used to study the efficacy and toxicity of CD19-targeted CAR T-cell therapy for B-cell malignancies¹¹ and evaluate the effects of combination therapies on tumor growth and immune cell infiltration.¹² However, it is important to note that there are limitations to these models, including the variability in engraftment efficiency, immune cell development¹³ and lineage skewing,¹⁴ which can affect the reproducibility of results,¹⁵ potential species-specific differences in immune function,^12,16 and the fact that these mice may not fully recapitulate the complexity and heterogeneity of the human immune system and the tumor microenvironment,¹⁷ all of which can limit the translational potential of findings from humanized mouse studies.

Although syngeneic mouse models lack human-specific interactions and the ability to study the efficacy of human-specific immunotherapies, they have several advantages over humanized mouse models. They are less variable and have a more predictable immune response because their immune system, the tumor cells, and the host tissue are genetically identical, allowing for more relevant interactions between the immune system and the tumor microenvironment, which is critical for understanding the mechanisms of action of immunotherapies. Other advantages of syngeneic mouse models are that immunotherapies can be studied in fully immunocompetent hosts with physiologically intact immune responses, and they are more cost-effective and easier to maintain than humanized mouse models. In conclusion, both models complement each other and provide a more comprehensive understanding of immunotherapy's mechanisms of action and potential side effects, helping to develop more effective and safer treatments for cancer.

The signaling lymphocytic activation molecule family member 7 (Slamf7) is a cell surface glycoprotein that is primarily expressed on immune cells, including natural killer cells, plasma cells, and subsets of T cells. Due to its robust and high-level expression on malignant plasma cells,¹⁸ SLAMF7 is a therapeutic target for the treatment of multiple myeloma.^19,20,21,22 We therefore selected murine Slamf7 (19A, CD319, CRACC, and CS1) as a model antigen for the direct comparison of several modalities of immunotherapy in a syngeneic mouse model of multiple myeloma. Furthermore, these therapeutic approaches could also be evaluated in the setting of allogeneic hematopoietic stem cell transplantation (aHSCT).

Dark Agouti rats were immunized with recombinant mouse Slamf7 extracellular domain to generate a rat mAb against mouse Slamf7. Rapid amplification of complementary DNAs ends polymerase chain reaction identified the variable heavy and light sequences of positive hybridoma clones.^23,24 The resulting mAb was designated as Slamf7-mAb. A bispecific antibody against CD3ε and Slamf7, Slamf7-BiTE, was generated by cloning the single-chain variable fragment (scFv) of the anti-CD3ε antibody CRL-145 with a glycine-serine linker along with the previously identified scFv of the α-Slamf7 antibody.^25,26 Slamf7-BiTE constructs were produced in Freestyle CHO-S cells and purified.

James Kochenderfer and Steven Rosenberg donated the CAR CD19 plasmid.²⁷ The Slamf7-CAR was created by combining the α-Slamf7 scFv with msCD8α's hinge and transmembrane domain, as well as intracellular signaling sequences from msCD28, 4-1BB, and CD3ζ.²⁸ This was cloned into the same murine stem cell virus retroviral vector used for CAR CD19. Mouse CD3⁺ T cells were transduced following standard protocols.²⁹ Phoenix-Eco cells were transfected with DNA using FugeneHD, and transfection was confirmed the next day using pEGFP-N1 as a reporter. CD3/CD28-coated plates were used to activate CD3⁺ T cells. RetroNectin-coated plates were used to transduce activated T cells with the retrovirus-containing supernatant from transfected Phoenix-Eco cells. The T cells were then cultured for 3 to 4 days in T-cell base medium supplemented with human interleukin (hIL)-7 and hIL-15. Fluorescence-activated cell sorting (FACS) analysis was used to detect CART⁺ cells.

BALB/c and CB6F1 hybrid mice obtained from Charles River (Sulzfeld, Germany) were housed in the Medical Faculty's animal facility in Muenster, Germany, under specific pathogen-free conditions. Female mice used for experiments were aged ∼12 to 20 weeks, and those used for T cell isolation were aged around 4 to 8 weeks. The regional governmental authorities approved all procedures in accordance with European regulations. The BALB/c-derived plasmacytoma cell line MPC-11 (ATCC CCL-167)³⁰ was obtained from the American Type Culture Collection (Manassas, VA). MPC-11 wild type (wt) naturally expressed Slamf7, whereas the MPC-11 knockdown (kd) cell line expressed low levels of Slamf7 and was cultured under the same conditions as the wt cell line.

The Cas9-Puromycin vector (GenScript, L00693) was used to clone guide RNA primers that targeted Exon 2 and 3 of mouse Slamf7. MPC-11 cells were then transfected with the guide RNA primers using the FugeneHD transfection reagent (Promega, E2311) following the company’s protocol. After transfection, the cells were selected using puromycin (invivoGen, ant-pr-1) for several weeks. Clones obtained from single-cell dilution that expressed Slamf7 at lower levels than MPC-11 wt were identified using flow cytometry (FACS) staining for Slamf7 expression.

In the naive mouse model, IV injections were administered immediately after tumor inoculation. MAb treatment involved a dose of 10 mg per kg body weight (bw), whereas BiTE treatment comprised 1.25 mg per kg bw given twice a week. For CART treatment, mice received 100 mg per kg body weight cyclophosphamide for lymphodepleting chemotherapy intraperitoneally, followed by a single application of CART (10e6) on the next day, and tumor injection 1 day later. Phosphate-buffered saline (PBS) injections served as controls. In the transplant setting, female CB6F1 (H-2Kbxd) mice received total body irradiation of 9 Gy 1 day before transplant. Bone marrow cells from donor female BALB/c (H-2 dissociation constant [Kd]) mice were administered via tail vein at 1e6/g bw. Tumor injection and immune therapy were initiated ∼3 weeks after transplant.

For in vitro cell cytotoxicity, MPC-11 wt and kd cell lines were labeled with BioTracker 488 Membrane Dye. Pan T cells were isolated from BALB/c spleens and mixed with tumor cells in a 10 to 1 effector-to-target ratio. The cell mixture was plated in a non-TC treated 96 U-well plate in assay medium. Antibodies were added to the cells at specific concentrations, and Slamf7-mAb was used to block T cells in some experiments. After 48 hours of incubation, cells were stained and analyzed using FACS.³¹ Specific cytotoxicity was calculated based on live target cell counts, and T-cell activation was determined using CD69 expression. For antibody-dependent cellular cytotoxicity (ADCC) analysis, the mouse FcyRIV ADCC bioassay from Promega was used according to the manufacturer's protocol.

We initially generated novel rat mAb targeting Slamf7, resulting in the generation of 3 distinct clones (3.1.4, 49.1.1, and 12.3.1). Among these clones, clone 3.1.4, belonging to the immunoglobulin G2 (IgG2) isotype, was chosen for further investigations due to its superior binding affinity to Slamf7 compared with other clones (supplemental Figure 1). To study effector functions in murine models and to avoid cross-species reactivity, we chimerized the antibody by switching the invariable regions of the heavy and light chains of the rat Fc-IgG2a domain with murine Fc-IgG2a (Figure 1A,I). We confirmed the integrity of the purified antibody (Slamf7-mAb) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed intact heavy (50 kDa) and light chains (25 kDa) compared with an isotype control (Figure 1B,I). Slamf7-mAb bound to Slamf7 endogenously expressed by the mouse myeloma cell line MPC-11 (MPC-11 wt), and no binding was observed after the Slamf7 gene had been knocked out³² in MPC-11 cells (MPC-11 kd) using CRISPR-Cas9 (Figure 1C).

To generate a bispecific antibody against murine Slamf7 and murine CD3 (Slamf7-BiTE), we fused the scFv of the Slamf7 antibody (3.1.4) to the scFv of an antibody that recognizes the murine CD3ε chain (clone 145-2C11) using a glycine-serine linker (Figure 1D,I).²⁵ Purity of Slamf7-BiTE was confirmed by western blot using a carboxy terminus myc-tag (Figure 1E). Specific binding of Slamf7-BiTE was confirmed by staining of MPC-11 wt (Figure 1F, left) but not MPC-11 kd (Figure 1F, middle) and binding to CD3⁺ T cells compared with isotype control (Figure 1F, right). The anti-CD3 scFv used in our constructs was derived from the 145-2C11 antibody, a hamster anti-mouse CD3ε antibody with relatively low affinity (Kd, ∼7 × 10^–8 M),^25,33,34,35 which has previously been used in functional BiTE constructs.³¹ The efficacy and toxicities of antibody-based immunotherapies can be influenced by the individual binding affinities of the antigen-binding fragment. To address this, we conducted Biacore analyses of the Slamf7-mAb, resulting in a Kd of 6.97 × 10^–10 M, and Slamf7-BiTE, with a slightly lower Kd of 6.64 × 10^–9 M (supplemental Figure 2).

To obtain a CAR that specifically recognizes Slamf7, the variable regions of the hybridoma 3.1.4 light and heavy chains were cloned into a retroviral plasmid encoding the transmembrane domain of murine CD8α as well as the intracellular signaling sequences from murine CD28, 4-1BB, and CD3ζ (Figure 1G,I). We identified anti-Slamf7-CAR–transduced T cells (Slamf7-CART) by staining for scFv (Figure 1H). Transduction efficiencies varied between 25% and 40% (Figure 1H).

To assess the cytotoxic effectiveness in vitro, we used MPC-11 wt cells that express Slamf7 (Figure 1C) as a target. At effector-to-target ratios ranging from 40:1 to 1:1, Slamf7-BiTE induced T cells to kill >80% of MPC-11 wt targets (Figure 2A, solid black). This effect was specific and required the expression of Slamf7 by target cells, because cell lysis was reduced to <40% and <4% at ratios of 40:1 and 1:1, respectively, when MPC-11 kd cells were used as targets (Figure 2A, dotted black). Therefore, Slamf7-BiTE increased the cytotoxicity of T cells by more than 20-fold at low effector-to-target ratios of 1:1, and even at very high ratios of 40:1, this increase remained higher than twofold and was statistically significant (P = .007). When an irrelevant BiTE was used, we observed very little T-cell–mediated cytotoxicity, which remained <13% regardless of whether the target cells expressed Slamf7 (Figure 2A, solid gray; Figure 2A, dotted gray). There was no statistically significant difference when Slamf7-BiTE–induced lysis of MPC-11 kd was compared with using an irrelevant BiTE and MPC-11 wt (P = .1032) or MPC-11 kd (P = .1110). At a constant 10:1 effector-to-target cell ratio, the Slamf7-BiTE–induced cytotoxicity of MPC-11 wt increased in a dose-dependent manner, showing a sigmoid slope with a half maximal target cell lysis (EC50) at a concentration of ∼0.0247 μg/mL (Figure 2B, filled circles). No dose dependency was observed when MPC-11 kd targets (Figure 2B, filled squares) or irrelevant bispecific T-cell engager (irrBiTE) were used (Figure 2B, gray symbols), highlighting the requirement of Slamf7 expression by target cells for specific Slamf7-BiTE–mediated killing by T cells.

To exclude a relevant impact of Slamf7-mediated inhibitory signaling in Slamf7-BiTE–mediated activation, we blocked self-ligation of Slamf7 by preincubating effector T cells with an antibody directed against Slamf7 before measuring cytotoxicity (Figure 2C, solid gray). There was no significant difference (P = .6995) in cytotoxic activity at BiTE concentrations ranging from 0.021 to 15 μg/mL with (Figure 2C, solid gray) or without (Figure 2C, solid black) blocking of Slamf7 on T cells. As a control, incubation of T cells with monoclonal Slamf7 antibody alone did not result in any appreciable target cell lysis (Figure 2C, dotted gray).

To further assess the activation of T cells through the α-CD3 arm, the expression of CD69 was evaluated 48 hours after incubation with Slamf7-BiTE (Figure 2D, solid bars). Blocking Slamf7 on T cells (Figure 2D, solid gray) slightly increased CD69 expression levels compared with unblocked T cells (Figure 2D, solid black). This effect was small but statistically significant (P = .037) across a wide range of BiTE concentrations.

We used Jurkat cells with a luciferase reporter transgene that is activated upon binding of antibody to the murine FcγRIV receptor to evaluate the ability of Slamf7-mAb on ADCC. Slamf7-mAb induced luminescence beginning at concentrations of 0.016 μg/mL, reaching near maximal luminescence at 0.4 μg/mL, after which the signal reached a plateau at about 60 000 relative light units in the presence of MPC-11 wt target cells (Figure 2E, solid upright triangles). In the presence of MPC-11 kd luminescence remained <4500 relative light units even at the highest assessed antibody concentration of 10 μg/mL (Figure 2E, solid inverted triangles). No appreciable ADCC using luminescence as a surrogate parameter could be detected when an isotype control antibody (Figure 2E, circles) or Slamf7-BiTE (Figure 2E, squares) was incubated with MPC-11 wt or kd targets.

To achieve sufficient transduction efficiencies of ∼20% to 30%, freshly harvested T cells had to be activated using plate-bound antibodies directed against CD3 and CD28. Cytotoxicity of resulting Slamf7-CART was measured in a similar assay used to evaluate Slamf7-BiTE (Figure 2A). Slamf7-CART lysed ∼45% of MPC-11 wt target cells at an effector-to-target ratios of 1:1 and >90% at 40:1 (Figure 2F, solid black bars). Cytotoxicity against Slamf7-expressing targets was significantly (P < .01) higher than that of MPC-11 kd cells expressing very little Slamf7 (Figure 2F, shaded black bars) or when irrelevant CAR T cells recognizing murine CD19, which is not expressed by MPC-11 cells (data not shown), were used (Figure 2F, solid and shaded black bars).

To compare the effects of different antibody-mediated immunotherapeutic formats on tumor regression in vivo, female BALB/c (H-2Kd) mice were challenged with the syngeneic BALB/c-derived myeloma cell line MPC-11 wt (H-2Kd) by injection into the right flank. Starting the same day mice received different doses of Slamf7-mAb and Slamf7-BiTE twice weekly (Figure 3A, bottom). For lymphodepletion before CART, mice received cyclophosphamide on day –2 and Slamf7-CART on day –1. Challenge with MPC-11 wt followed on day 0 as above (Figure 3A, top). In all experiments, tumor growth, bw, and general wellbeing of mice were assessed daily until experiments ended on day 14. Both, Slamf7-mAb and Slamf7-BiTE significantly reduced tumor growth compared with PBS control injections (P < .5). The observed inhibition of tumor growth was consistently more pronounced with Slamf7-BiTE than with Slamf7-mAb, although this difference was not statistically significant. (Figure 3B). Although Slamf7-mAb did not affect animal health and bw, Slamf7-BiTE caused significant bw loss of health. The undulating pattern of weight loss was correlated with individual injections of Slamf7-BiTE (Figure 3C). Typically, animals started to recover 24 hours after BiTE injection and regained their starting bw ∼3 days later (Figure 3C).

Compared with control, Slamf7-CART led to retardation of tumor growth (P > .1). Even nontransduced T cells had some effect on tumor growth, but this was less pronounced (Figure 3D). Although all mice showed some degree of transient weight loss between days 1 and 5 after lymphodepletion, this was most evident in mice receiving Slamf7-CART (Figure 3E). CART cells expansion was documented by flow cytometry of the peripheral blood, in which CD3⁺ T cells expressing scFv Slamf7 could readily be detected (Figure 3F).

Among hematological malignancies, relapse remains the primary cause of treatment failure after allogeneic transplantation.^36,37 To develop more effective therapeutic strategies for these patients, experimental models play a crucial role. In this study, we investigated the efficacy of different antibody-based immunotherapeutic formats in the context of aHSCT. For this purpose, we used a nonlethal parent-into-irradiated F1 model, which allows for continuous monitoring and intervention after transplantation.³⁸ In this model, very mild graft-versus-host disease results from partial major histocompatibility complex mismatch between H-2b haplotype donor cells and lethally irradiated H-2b/d haplotype recipients. Bone marrow from mice on the Balb/c background was transferred into lethally irradiated CB6F1 recipients on day 0 (Figure 4A, top left). Three weeks after transplant, mice received lymphodepleting chemotherapy, Slamf7-CART, and the tumor challenge on 3 consecutive days (Figure 4A, top right) or an injection with Slamf7-mAb or Slamf7-BiTE followed by tumor injection on the same day (Figure 4A, bottom right). In all experiments, tumor growth, bw, and general wellbeing of mice were assessed daily. To document engraftment and complete donor cell chimerism, we quantified the number of H-2b⁺ and H-2d⁺ cells in the peripheral blood by flow cytometry 2 weeks after aHSCT (Figure 4B). Successful transfer and expansion of Slamf7-CART was assessed at the end of each experiment by analyzing individual blood samples using flow cytometry and staining for CD3 and scFv Slamf7 (Figure 4C). The CAR of Slamf7-CART could only be detected in mice that received CAR T cells (Figure 4C, III). Treatment with Slamf7-mAb (Figure 4D, triangles) and Slamf7-BiTE (Figure 4D, filled squares) delayed tumor progression in this allogeneic transplantation model compared with PBS and irrBiTE control groups (Figure 4D, open squares and circles, respectively), as had been observed in the nontransplant setting (Figure 3B). Although animals that had received irrBiTE and Slamf7-mAb showed some degree of bw loss (Figure 4E, triangles and circles), the more pronounced and undulating weight loss in correlation to the administration of BiTE that we had observed before (Figure 3C) was only evident in mice that had been treated with Slamf7-BiTE (Figure 4E, filled squares). Unlike the animals in the nontransplant setting (Figure 3C,E), mice that had been treated with Slamf7-mAb, Slamf7-BiTE, or irrBiTE did not fully recover their bw by the end of the experiment (Figure 4E). No appreciable weight loss was detectable in control mice that had only received PBS injections (Figure 4E, open squares). Treatment of animals that had undergone aHSCT with Slamf7-CART significantly (P = .152) reduced tumor growth (Figure 4F, diamonds) and had no relevant impact on bw (Figure 4G), compared with control animals.

Slamf7 is a self-associating member of the signaling lymphocytic activation molecule family that is evolutionary conserved across mammals and is expressed on cells of the immune system. Because of its high expression in multiple myeloma, it is a clinically relevant target in the treatment of patients with relapsed and refractory multiple myeloma. We have developed several immunotherapeutic approaches that use the identical complementarity-determining regions derived from a single mAb that recognizes murine Slamf7 (Figure 1). Using the myeloma cell line MPC-11 as a target, we performed comparative side-by-side analyses of a mAb, a BiTE, and CART under controlled experimental conditions in immunocompetent syngeneic hosts (Figures 2 and 3). In these model systems, antitumor effects could not only be studied in unmanipulated mice in the steady state (Figures 2 and 3) but also in mice that had previously received an allogeneic stem cell transplant (Figure 4). These results are meaningful because both, antitumor as well as side effects are modulated in a meaningful way after a stem cell transplant. Certain immunotherapies regularly cause detrimental side effects such as cytokine release syndrome (CRS) as well as immune effector cell-associated neurotoxicity syndrome that despite appropriate treatment can be life threatening in humans. Crucially, our models mimic these side effects by not only causing general malaise but also objective weight loss in test animals.

Previous studies have established that IV administration of the mAb 145-2C11, which was used in the construction of Slamf7-BiTE, can induce clinical and laboratory manifestations reminiscent of those observed in patients suffering from CRS. These manifestations include weight loss, hepatosplenomegaly, thrombocytopenia, increased vascular permeability, lung inflammation, and hypercytokinemia. Additionally, the investigation conducted in this regard involved the assessment of blood cytokine levels and gene expression in various tissues, such as lung, liver, and spleen, revealing significant upregulation of proinflammatory cytokines, including interleukin-2, interferon gamma, tumor necrosis factor-α, and interleukin-6, which preceded the onset of clinical CRS symptoms.³⁹ Although we have not independently confirmed these findings in this study, it is tempting to speculate that the strikingly similar symptoms observed after the administration of Slamf7-BiTE may also be indicative of CRS. Future studies using this model system should aim to address this hypothesis and further elucidate the underlying mechanisms.

SLAM receptors triggered by homotypic or heterotypic cell-cell interactions are modulating the activation and differentiation of a wide variety of immune cells, and Slamf7 has been identified as a negative regulator of T-cell activation.⁴⁰ Neither Slamf7-BiTE–mediated cytotoxicity nor the upregulation of the early T-cell activation marker CD69 after the engagement of the T-cell receptor by Slamf7-BiTE appeared to be affected significantly when we blocked self-ligation of Slamf7 by preincubating effector T cells with an antibody against Slamf7. This indicated that any inhibitory functions of Slamf7 had no relevant impact on cytotoxicity and T-cell activation in our model.

All therapeutic approaches showed significant antitumor activity in vitro as well as in vivo, but we did not observe complete tumor eradication under these experimental settings.

Although phenomena such as clonal exhaustion of effector T cells during chronic antigen exposure could contribute to this finding,⁴¹ it is conceivable that reducing the number of injected cancer cells might mimic minimal residual disease better and hence allow for better tumor control or even eradication.^42,43 Further studies incorporating tumor heterogeneity and niche interactions are needed for a comprehensive minimal residual disease model. This study evaluated different antibody formats targeting the same antigen in a syngeneic model. We selected a representative format from chimeric mAbs, bispecific formats similar to blinatumomab, and CART constructs, all effective in mouse models.^28,31,44 

Clone 3.1.4 was selected for its intermediate affinity to Slamf7 to avoid off-target effects or rapid clearance. The BiTE construct used the CD3 arm (clone 145-2C11) for its specific recognition of mouse CD3ε and efficacy in T-cell activation. Amino terminus placement of the CD3 arm was chosen for superior T-cell activation.^25,31 The CART construct used a third-generation design, chosen for its effectiveness in preclinical mouse models, although not yet clinically approved.²⁸ 

Although the 3 immunotherapeutic modalities chosen for this study constitute just a fraction of the available immunotherapeutic options, our model serves as a valuable instrument for enhancing the understanding and optimization of antitumor effects, particularly those influenced by structural variations. Moreover, the flexibility of our model allows for straightforward modifications to investigate and contrast other clinically pertinent constructs. This includes the exploration of second-generation CAR constructs with fewer signaling domains that have already been approved for clinical use.⁴⁵ 

The study showed modest effects on tumor eradication, even in early stages, especially with established tumors (data not shown). Despite limitations, it lays a valuable foundation for future studies to explore various antigens, constructs, and tumor entities, expanding on the framework presented.

A subcutaneous tumor model was chosen for its reproducibility and quantifiable results, ideal for comparing 3 immunotherapies and assessing their effects on tumor growth and toxicity. However, this model does not fully replicate multiple myeloma, particularly the bone marrow niche crucial for immunotherapy.^46,47 Future research needs models that more accurately represent multiple myeloma and include preestablished tumor models to better understand the disease and treatment efficacy.⁴⁸ This will improve the evaluation of new therapies for multiple myeloma.

Although treatment options for patients with myeloma have improved significantly over the past decades, myeloma remains an incurable malignancy for the vast majority of patients.^49,50 Other targets besides SLAMF7 have been discovered, such as the B-cell maturation antigen (BCMA), which represents another promising target for treating myeloma. BCMA is highly expressed in multiple myeloma, and various immune therapeutics targeting BCMA have been developed, including the mAb-drug conjugate belantamab mafodotin as well as the BCMA-directed CART therapies, idecabtagene vicleucel and ciltacabtagene autoleucel, that have both been approved for multiple myeloma therapy. Although Slamf7 might not have the molecular prerequisites to be a suitable target for an ADC, our model system could be adapted by using a mAb as a basis that is more capable of internalizing ADC complexes.

Teclistamab is a recently approved bispecific antibody that targets the CD3 receptor and BCMA.⁵¹ Similar to other T-cell–mediated therapeutics, ∼72% of patients experience CRS after receiving teclistamab. Although preclinical studies using xenograft models inoculated with human myeloma and T cells demonstrated that teclistamab had antitumor activity, they failed to flag adverse symptoms such as CRS.⁵² Although relevant side effects can also be missed in syngeneic animals models as the infamous phase 1 study of the superagonistic anti-CD28 antibody TGN1412 has shown,⁵³ they can be useful in complementing efficacy data derived from xenograft models. The preserved interactions between immune cells and host tissues in syngeneic models provide valuable insights into the development of CRS, immune effector cell-associated neurotoxicity syndrome, or surrogate symptoms such as malaise or weight loss. These models play a crucial role in identifying potential adverse effects early in the drug development process and offer valuable opportunities to study and better understand undesired side effects.⁵⁴ 

A preclinical in vivo model that is capable of assessing the impact of immune therapeutics on both tumor growth and potential adverse side effects resulting from the interactions between the treatment and the host’s immune system is urgently required. Although current preclinical models, such as xenograft/humanized models, are effective at demonstrating efficacy against human tumor cells, they lack a functioning immune system and syngeneic host tissue, which fails to accurately reflect the complex interactions between the treatment and the host organism. Our newly developed model of hematological disease allows for side-by-side comparisons of different immunotherapies while using a syngeneic and functioning immune system. Furthermore, our model accurately recapitulates the desired antitumor activity of individual agents as well as adverse effects regularly observed with these therapies, such as CRS and neurotoxicity. These attributes will allow for further studies to better understand how desired antitumor activity and detrimental side effects are intertwined at the cellular and molecular level. Therefore, although more research is warranted to corroborate the link between clinical symptoms and CRS as well as to test the activity of immunotherapies against other tumor entities and antigens, our model represents a valuable basis for preclinical tools that can be adapted for the development and study of a wide variety of immunotherapeutic treatment modalities.

Contribution: K.F., J.C.A., and M.S. designed the study; K.F., A.W., and C.S. performed the key experiments; and K.F., J.C.A., A.W., C.S., S.H., G.L., and M.S. interpreted the data and wrote and rewiewed the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Matthias Stelljes, Department of Medicine A, Hematology, Oncology, and Pneumology, University Hospital Münster, Albert-Schweitzer-Campus 1, 48149 Münster, Germany; email: stelljes@ukmuenster.de.