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Simple Summary CD3-bispecific antibody therapy is a form of immunotherapy that enables soldier cells of the immune system to recognize and kill tumor cells. This type of therapy is currently successfully used in the clinic to treat tumors in the blood and is under investigation for tumors in our organs. The treatment of these solid tumors faces more pronounced hurdles, which affect the safety and efficacy of CD3-bispecific antibody therapy. In this review, we provide a brief status update of this field and identify intrinsic hurdles for solid cancers. Furthermore, we describe potential solutions and combinatorial approaches to overcome these challenges in order to generate safer and more effective therapies. Abstract Immunotherapy of cancer with CD3-bispecific antibodies is an approved therapeutic option for some hematological malignancies and is under clinical investigation for solid cancers. However, the treatment of solid tumors faces more pronounced hurdles, such as increased on-target off-tumor toxicities, sparse T-cell infiltration and impaired T-cell quality due to the presence of an immunosuppressive tumor microenvironment, which affect the safety and limit efficacy of CD3-bispecific antibody therapy. In this review, we provide a brief status update of the CD3-bispecific antibody therapy field and identify intrinsic hurdles in solid cancers. Furthermore, we describe potential combinatorial approaches to overcome these challenges in order to generate selective and more effective responses.

Background The tumor-associated antigen 5T4 is expressed across a wide range of solid cancers. DuoBody-CD3x5T4 is a bispecific antibody (bsAb) that crosslinks CD3 on T cells with 5T4 on tumor cells, thereby inducing T-cell activation and T-cell mediated cytotoxicity in 5T4-expressing tumor cells. Here, we tested the capacity of DuoBody-CD3x5T4 to engage different T-cell subsets in vitro and investigated the mechanism of action (MoA) in vivo by combining preclinical efficacy studies with exploratory pharmacodynamic (PD) biomarker analyses Methods Immunohistochemistry was performed on patient-derived tumor tissue-microarrays using a commercial 5T4 monoclonal antibody (EPR5529). The capacity of DuoBody-CD3x5T4 to engage naive and memory T-cell subsets was assessed in co-cultures of T cells and 5T4-positive tumor cells, using T-cell activation and T-cell mediated cytotoxicity as readouts. Anti-tumor activity in vivo as well as peripheral and intratumoral PD biomarkers were investigated in humanized mice bearing 5T4-expressing cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) tumor models. Results High prevalence of 5T4 expression (in >86% of biopsies) was observed in NSCLC, SCCHN, TNBC, bladder, esophageal, prostate and uterine cancer. In co-cultures of 5T4+ tumor cells and T cells in vitro, DuoBody-CD3x5T4 induced dose-dependent cytotoxicity, associated with T-cell activation, proliferation, and cytokine, perforin and granzyme production. Crosslinking of T cells with 5T4-expressing tumor cells was essential as no cytotoxicity was observed in CRISPR-Cas9-generated 5T4-knockout tumor cells or with control bsAbs targeting only CD3 or 5T4. Importantly, naive and memory CD4+ or CD8+ T-cell subsets had equal capacity to mediate DuoBody-CD3x5T4-induced cytotoxicity, although naive T-cell subsets showed slower kinetics. DuoBody-CD3x5T4 (0.5–20 mg/kg) demonstrated anti-tumor activity in 5T4+ breast and prostate cancer CDX and lung cancer PDX models in humanized mice. Treatment with DuoBody-CD3x5T4 was associated with intratumoral and peripheral T-cell activation as well as elevated cytokine levels, including IFNγ, IL-6 and IL-8, in peripheral blood. Conclusions DuoBody-CD3x5T4 induced T-cell mediated cytotoxicity in 5T4-expressing tumor cells, associated with T-cell activation and cytokine production in vitro. DuoBody-CD3x5T4 efficiently engaged naive and memory T cells within both CD4+ and CD8+ T-cell populations to induce T-cell mediated cytotoxicity in 5T4+ tumor cells. In humanized CDX and PDX mouse models, DuoBody-CD3x5T4 showed anti-tumor activity, in addition to PD biomarkers associated with T-cell activation in the tumor and periphery. Currently, DuoBody-CD3x5T4 is being investigated in a first-in-human clinical trial for the treatment of solid tumors (NCT04424641), in which exploratory biomarker analyses to study the clinical MoA and PD are included. Ethics Approval The CDX animal experiments performed are in compliance with the Dutch animal protection law (WoD) translated from the directives (2010/63/EU) and are approved by the Ethical committee of Utrecht. For the PDX models, all patients had given written informed consent, and the animal experiments were carried out in accordance with the German Animal Protection Law (LaGeSoBerlin, A0452/08). The studies were approved by the local Institutional Review Board of Charite University Medicine, Germany.

Background: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. Methods: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. Findings: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. Interpretation: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. Funding: Genmab Keywords: Bispecific antibody, CD3, CD20, T cell redirection, B cell malignancy, Subcutaneous administration

Evers et al. employed CRISPR/Cas9 technology to generate B cell populations missing the BCR as an additional (negative) control, to investigate the contribution of the BCR in anti-CD20–mediated complement-dependent cytotoxicity (CDC) that we proposed ([1][1]). We agree with the notion that loss of

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic. Copyright © 2016 by The American Association of Immunologists, Inc.

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab–CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb–CD20 complexes by the B cells themselves, with internalization of ∼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75–90% in

DuoBody®-CD3xCD20 (GEN3013) is a bispecific antibody (bsAb), recognizing the T-cell antigen CD3 and the B-cell antigen CD20, that triggers potent T-cell-mediated lysis of CD20-expressing cells. DuoBody-CD3xCD20 is a full-length bispecific IgG1 generated by controlled Fab-arm exchange (cFAE) [1, 2] and contains an effector function-silenced Fc region. In vitro, DuoBody-CD3xCD20 induced potent activation, proliferation and cytotoxic activity of both CD4+ and CD8+ T cells in the presence of CD20-expressing cells, as measured by flow cytometry and bromodeoxyuridine (BrdU) incorporation assays. DuoBody-CD3xCD20 induced T-cell-mediated cytotoxicity towards a diverse panel of cell lines derived from various B-cell malignancies and endogenous B cells, with EC50 values in the low picomolar range (EC50: 0.2-5.0 pM). The CD20-specific antibody 7D8 [3-5] forms the basis for the CD20-specific Fab arm of DuoBody-CD3xCD20. To study the contribution of this specific Fab arm to the observed potency of DuoBody-CD3xCD20, we compared the target binding characteristics and the capacity to induce T-cell-mediated cytotoxicity of a CD3 bsAb based on 7D8, with CD3 bsAbs using B-cell targeting arms derived from alternative CD20 antibodies or from antibodies against other well-known B-cell membrane molecules CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR. In addition, target expression levels of the B-cell targets were assessed in a panel of B-cell lines. Using a classic chromium release assay, the 7D8-based CD3 bsAb displayed cytotoxic activity superior to all other B-cell-targeting CD3 bsAbs tested, including alternative CD20-targeting CD3 bsAbs. This unique cytotoxic activity could not be explained by expression levels of the target antigen, nor by the binding affinity or epitope of the B-cell specific Fab arm. This illustrates the complexity of factors that determine the potency of CD3 bsAbs. The anti-tumor activity of DuoBody-CD3xCD20 was confirmed in vivo in humanized mouse models using three different B-cell lymphoma xenograft models, in prophylactic and therapeutic settings. Non-clinical safety studies with DuoBody-CD3xCD20 in cynomolgus monkeys demonstrated profound and long-lasting B-cell depletion (at least 70 days, at dose levels > 0.1 mg/kg) from both peripheral blood and lymphoid organs. B-cell depletion was reversible, with time to B-cell recovery correlating with the treatment dose. Notably, at the same dose level, B-cell depletion was comparable between subcutaneous and intravenous administration. Pharmacokinetic (PK) analysis demonstrated comparable bioavailability for the two administration routes, although peak plasma levels were lower and delayed after subcutaneous administration. Moreover, lower plasma cytokine levels were observed after subcutaneous administration. Based on these data, Genmab has initiated a First-in-Human clinical trial to evaluate the safety and preliminary efficacy of DuoBody-CD3xCD20 by subcutaneous administration in patients with B-cell malignancies. The study is currently enrolling (EudraCT No: 2017-001748-36). References Labrijn, A.F., et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A, 2013. 110(13): p. 5145-50. Labrijn, A.F., et al., Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc, 2014. 9(10): p. 2450-63. Teeling, J.L., et al., Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood, 2004. 104(6): p. 1793-800. Teeling, J.L., et al., The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20. Journal of Immunology, 2006. 177(1): p. 362-71. van Meerten, T., et al., HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis. Haematologica, 2010. 95(12): p. 2063-71. Disclosures Hiemstra: Genmab: Employment, Other: Warrants. Engelberts:Genmab: Employment, Other: Warrants. de Jong:Genmab: Employment, Other: Warrants. Schuurhuis:Genmab: Employment, Other: Warrants. Salcedo:Genmab: Employment, Other: Warrants. Verploegen:Genmab: Employment, Equity Ownership. van der Zee:Genmab: Employment, Other: Warrants. Gerritsen:Genmab: Employment, Other: Warrants. Losic:Genmab: Employment, Other: Warrants. Horbach:Genmab: Employment, Other: Warrants. Oliveri:Genmab: Employment, Other: Warrants. Lammerts van Bueren:Genmab: Employment, Other: Warrants. Autzen Usher:Genmab: Employment, Other: Warrants. Schuurman:Genmab: Employment, Other: Warrants. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Breij:Genmab: Employment, Equity Ownership.

T-cell redirecting therapy has taken a prominent area in immuno-oncology. T cells driven by a tumor-specific antigen, traffic into the tumor and initiate tumor killing. However, this is often hampered by inhibitory factors in the tumor microenvironment. In recent years, researchers have been actively developing T-cell redirecting bispecific antibodies, which binds to a specific tumor associated antigen (TAA) on tumor cells and CD3 (usually epsilon chain, CD3E) on T cells. This physically links the tumor cell to the T-cell which leads to MHC-independent recognition and killing of cells carrying the TAA. Several CD3 recruiting bispecific antibodies that have been approved are now in clinical trials and demonstrate promising efficacy. However, research on new therapeutic T-cell redirecting antibodies is often hampered by a lack of proper in vivo models, due to the absence of cross-reactivity with mouse CD3ε. We have now built a human CD3E BAC transgenic model in BALB/c background to address this issue. These transgenic mice express both human- and mouse CD3ε in more than 80% of the T cells. Unlike previously developed transgenic lines, where early T lymphocyte and natural killer cell development were blocked in mice with high copy numbers of the human CD3ε gene, our model is phenotypically normal with levels of T , B , and NK cells comparable to those in wild-type BALB/c mice. In an ex vivo T-cell stimulation assay, spleen-derived T cells could be activated by either anti-human CD3 antibody (OKT3) or anti-mouse CD3 antibody (145-2C11), indicated by significantly elevated CD25+/CD69+ population, as well as IL-2 and IFNγ release. In an efficacy assay, we inoculated syngeneic mouse CD20-expressing A20 lymphoma cells into the transgenic mice and treated with mCD20xmCD3 or mCD20xhCD3 bispecific antibodies, containing a human- or mouse CD3ε-specific CD3 arm, respectively. We found complete depletion of peripheral B cells 48 hours after dosing of either bispecific antibody. Treatment also induced 33% and 39% tumor growth inhibition at day 10, following 3 doses of 1 mg/kg mCD20xhCD3 and mCD20xmCD3 bispecific antibody, respectively. Taken together, our human CD3ε transgenic mice offer a novel model to assess the preclinical in vivo efficacy of human CD3-T-cell redirecting therapeutics. Citation Format: Mengmeng Yang, Mingkun Zhang, Sandra Verploegen, Patrick Engelberts, Yuxi Zhang, Lei Zheng, Keyi Zhu, Annie Xiaoyu An, Cunxiang Ju, Jing Zhao, Xiang Gao, Qian Shi, Davy Xuesong Ouyang. Establishment of a human CD3ε transgenic mouse model to assess anti-tumor efficacy of human T-cell-redirecting bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5669.

Clinical successes of antibody-based drugs has led to extensive (pre-) clinical development of human(ized) monoclonal antibodies in a great number of diseases. The high specificity of targeted therapy with antibodies makes it ideally suited for personalized medicine approaches in which treatments needs are tailored to individual patients. One aspect of patient stratification pertains to the accurate determination of target occupancy and target expression to determine individual pharmacodynamic properties as well as the therapeutic window. The availability of reliable tools to measure target occupancy and expression on diseased and normal cells is therefore essential. Here, we evaluate a novel human antibody detection assay (Human-IgG Calibrator assay), which allows the flow cytometric quantification of therapeutic antibodies bound to the surface of cells circulating in whole blood. This assay not only permits the determination of the number of specific antibody bound per cell (sABC), but, when combined with quantification of exogenously added mouse antibody, also provides information on binding kinetics and antigen modulation. Our data indicate that the calibrator assay has all properties required for a pharmacodynamic tool to quantify target occupancy of chimeric, humanized and human therapeutic antibodies during therapy, as well as to collect valuable information on both antibody and antigen kinetics.

To the editor: With great interest, we have read the article by Beers et al[1][1] documenting a potent B-cell depleting ability for type II (or tositumumab-like) CD20 antibodies. The authors conclude that complement-dependent cytotoxicity (CDC) is of little importance for B-cell depletion induced

A number of monoclonal antibodies (mAb) are presently in development or approved for CD20-directed immunotherapy. Ofatumumab, a human IgG1 anti-CD20 mAb, is currently being evaluated in phase III clinical trials for B-CLL and FL, and in phase II clinical trials for rheumatoid arthritis (RA). Rituximab, a chimeric IgG1 anti-CD20 mAb, has been approved for 1st line treatment of CD20-positive B cell lymphomas alone or in combination with chemotherapy, and in RA. Virtually all rituximab-treated patients relapse after single-agent treatment. The clinical efficacy of rituximab might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising anti-lymphoma effects. We studied the influence of statins on ofatumumab- and rituximab-mediated cytotoxicity. Surprisingly, B cells incubated with statins showed decreased rather than increased CD20 mAb-mediated complement-dependent cytotoxicity (CDC) in cell viability assays. However, cell lysis of statin-treated B cells remained higher when using ofatumumab in comparison to rituximab. Statins decreased CD20 immunostaining in flow cytometry but did not affect total cellular CD20 levels when compared to non-treated cells. Incubation of B cells with other cholesterol depleting agents (methyl-β-cyclodextrin (MβCD) and berberine) established that the presence of plasma membrane cholesterol and not lipid rafts may be required for CD20 mAb-mediated CDC. Cholesterol restitution reversed ofatumumab- and rituximab-mediated CDC and CD20 mAb staining. Statin incubation resulted in conformational changes of CD20 and impaired binding of CD20 mAb to the CD20 molecule as observed by atomic force microscopy. Freshly isolated cells of 4 B cell lymphoma patients were treated with the cholesterol-depleting agent MβCD, and CD20 mAb (B9E9) binding and rituximab-mediated CDC were significantly decreased (P