Dual-Affinity Re-Targeting Molecules Research Articles

CD3xCD19 DART molecule treatment induces non-apoptotic killing and is efficient against high-risk chemotherapy and venetoclax-resistant chronic lymphocytic leukemia cells

BackgroundBispecific antibodies are promising new therapeutics in B cell malignancies. Whether they lead to potent T cell activation despite described T cell dysfunction in chronic lymphocytic leukemia (CLL), and are...

CD3xCD19 Dart Treatment Is Efficient in Venetoclax Resistant CLL and Reverses T Cell Dysfunction

Intro - Agents targeting the apoptosis pathway, like the Bcl-2 inhibitor venetoclax, are highly effective in chronic lymphocytic leukemia (CLL). However, not all patients experience deep responses and acquired resistance has already been described. T cell mediated lysis is another tool currently exploited in hematologic malignancies. In contrast to acute lymphoblastic leukemia (ALL) however, efficacy of autologous based T cell therapy, such as CAR T cells, in CLL has been low. This is linked to a CLL mediated acquired T cell dysfunction. Bispecific T cell engagers targeting CD19 are successfully applied in ALL, but whether it overcomes the acquired T cell dysfunction in CLL is unknown. We therefore tested efficacy of a CD3xCD19 Dual Affinity Re-Targeting molecule (DART) in CLL. Since it has been observed that bispecific antibodies can overcome deficient synapse formation in CLL (Robinson et al, 2018) and based on our assumption that T cell mediated lysis differs from venetoclax-mediated killing, we hypothesized that usage of a CD3xCD19 DART in CLL overcomes T cell dysfunction and will be effective against venetoclax resistant CLL. Methods - Co-culture of CLL derived or aged-matched healthy donor (HD) CD4+ and/or CD8+ T cells with (CD40 activated) primary CLL or CD19+ cell lines JeKo-1 or Ramos in presence of CD3xCD19 (JNJ-64052781), CD3xFITC, anti-CD3/28 antibodies was performed. R esults - JeKo-1 cells were highly sensitive to CD3xCD19 mediated HD T cell killing with close to 70% of lysis in a concentration of 10ng/mL using an E:T ratio of 4:1. In the same conditions, primary CLL cells proved sensitive for CD3xCD19 mediated HD T cell killing with 50% of lysis. Killing was observed irrespective of IGHV mutation or chemorefractory status. We next compared HD with CLL-derived T cells by measuring activation levels between direct TCR (anti-CD3/CD28) and CD3xCD19 stimulation. As described, TCR stimulation resulted in impaired CLL CD4+ and CD8+ T cell activation and proliferation when compared to HD. In contrast, treatment of CLL derived PBMCs with CD3xCD19 did not resulted in dysfunctional CLL-derived T cell responses (Fig 1A-C). Consistently, co-culture of CLL derived CD4+, CD8+ or a combination with either JeKo-1 or allogeneic CLL cells in the presence of CD3xCD19 resulted in significant cytotoxicity (Fig. 1D). In the allogeneic setting, cytotoxic capacity of CD4+ T cells was similar to their CD8+ counterparts. When targeting autologous CLL, a benefit was observed when both CD4+ and CD8+ T cells were present (Fig. 1D). We then studied whether venetoclax resistant CLL cells could be targeted by CD3xCD19 mediated T cell killing. Bcl-2 overexpressing Ramos were equally lysed in presence of the CD3xCD19 DART as their wildtype counterpart, indicating that Bcl-2 expression does not inhibit CD3xCD19 mediated cell death. Following CLL cell stimulation by CD40 ligation, anti-apoptotic Bcl-XL, Bfl-1 and Mcl-1 are highly induced (Thijssen et al., 2015) resulting in venetoclax resistance (Fig 1E). Nevertheless, CD40L stimulated CLL cells were as efficiently lysed upon CD3xCD19 treatment as unstimulated CLL. (Fig 1F). This indicates that an augmented apoptotic threshold does not impact efficacy of CD3xCD19. Further examination of the mechanism of CD3xCD19 mediated killing showed that lysis depended on granzymes, as blocking granule exocytosis prevented cell death. Independence of the mitochondrial apoptotic pathway was shown by equal sensitivity to CD3xCD19 mediated T cell lysis comparing BAX/BAK knockout Jeko-1 cells to the parental cell line. Also, caspase blockage did not inhibit cell death, pointing to apoptosis independent killing. In concordance, PARP cleavage could only be detected when caspase activity was not blocked. Conclusion - This is the first report describing reversal of CLL mediated T cell dysfunction by applying a CD3xCD19 DART. Furthermore, it shows that venetoclax resistant CLL can still be efficiently targeted by T cells, in a non-apoptotic fashion. These results imply that T cell mediated therapy could be used alongside venetoclax. Figure 1 Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding. van der Windt:Genmab: Employment. Kater:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells.

Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells.

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.

ABSTRACTWith over 3.5 billion people at risk and approximately 390 million human infections per year, dengue virus (DENV) disease strains health care resources worldwide. Previously, we and others established models for DENV pathogenesis in mice that completely lack subunits of the receptors (Ifnar and Ifngr) for type I and type II interferon (IFN) signaling; however, the utility of these models is limited by the pleotropic effect of these cytokines on innate and adaptive immune system development and function. Here, we demonstrate that the specific deletion of Ifnar expression on subsets of murine myeloid cells (LysM Cre+ Ifnarflox/flox [denoted as Ifnarf/f herein]) resulted in enhanced DENV replication in vivo. The administration of subneutralizing amounts of cross-reactive anti-DENV monoclonal antibodies to LysM Cre+ Ifnarf/f mice prior to infection with DENV serotype 2 or 3 resulted in antibody-dependent enhancement (ADE) of infection with many of the characteristics associated with severe DENV disease in humans, including plasma leakage, hypercytokinemia, liver injury, hemoconcentration, and thrombocytopenia. Notably, the pathogenesis of severe DENV-2 or DENV-3 infection in LysM Cre+ Ifnarf/f mice was blocked by pre- or postexposure administration of a bispecific dual-affinity retargeting molecule (DART) or an optimized RIG-I receptor agonist that stimulates innate immune responses. Our findings establish a more immunocompetent animal model of ADE of infection with multiple DENV serotypes in which disease is inhibited by treatment with broad-spectrum antibody derivatives or innate immune stimulatory agents.

Non-depleting down-modulation of human B-cells through inhibitory coupling of FcγRIIB (CD32B) and the BCR with bi-specific DART molecules with extended pharmacokinetics. (51.8)

Abstract We have previously demonstrated the ability of bi-specific Dual-Affinity ReTargeting (DART) molecules to coligate the inhibitory FcγRIIb (CD32B) receptor and the BCR component, CD79B, to inhibit B-cell activation and dampen autoimmunity (Veri et al, Arthritis. Rheum. 2010). Here we describe development and characterization of CD32BxCD79B DARTs with enhanced pharmacokinetic (PK) properties through incorporation of an albumin binding domain (ABD) or an Fc region. These second generation CD32BxCD79B DARTs, expressed in CHO cells and purified to homogeneity, demonstrated anticipated multi-specific binding properties and effectively reduced naïve and memory B-cell responses in both healthy subjects and SLE patients. Both the ABD and Fc- bearing DARTs displayed increased serum half-life resulting in prolonged ability to functionally block human B-cell responses resulting in reduced circulating IgM and IgG levels in mice engrafted with human PBMC. In contrast to rituximab, CD32BxCD79B DARTs did not induce B-cell depletion in a human fetal liver CD34+ HSC reconstituted mouse model. In summary, CD32BxCD79B DARTs with extended PK properties have been developed that inhibit human B-cell activation in the absence of depletion, forming the basis for an alternate B-cell intervention strategy for controlling autoimmune disease through convenient dosing, while avoiding the more general immune suppression associated with existing B-cell targeted strategies.

Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma

AbstractWe describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell–specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell–killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19− cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Therapeutic control of B cell activation via recruitment of Fcγ receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold

To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.

Abstract 5629: Application of a novel bispecific antibody-based scaffold for optimal redirected T-cell killing of cancer cells

Abstract Despite significant progress, improved treatment modalities are required to eradicate cancer. Cytotoxic T lymphocytes are among the most potent cell populations capable of killing target cells and considerable effort has been dedicated to developing strategies aimed at redirecting T cells toward cancer, including the generation of bispecific molecules capable of co-engaging a tumor target together with an activating receptor on T lymphocytes. Limitations of the existing technology, however, including shortcomings in manufacturability, stability, specificity and potency, have often hampered progress in harnessing the power of T cells with bi-specific molecules. We have applied a novel antibody-based scaffold, termed DART (Dual-Affinity Re-Targeting), to redirect T-cell killing toward tumor cells in the absence of cognate interactions. DART molecules are structurally comprised of a covalently linked “diabody” generated through co-expression in mammalian cells of two separate Fv-encoding chains. To evaluate the ability of the platform to redirect T-cell killing, DARTs were designed to target hematological malignancies by comprising a B-cell lineage specificity (CD19) together with T-cell recognition (CD3 or TCR), while a DART comprising specificities for the HER2 target and the TCR was used to evaluate applicability to solid tumors. All three DART molecules assembled efficiently and were evaluated in a series of redirected T-cell killing assays to evaluate potency and mechanistic aspects of cytotoxicity. Both the CD19xCD3 and CD19xTCR DART molecules demonstrated efficient co-ligation of B-cell lymphoma cells with human T cells and mediated equivalently potent redirected T-cell killing against a panel of CD19-positive B-cell lymphoma cell lines but not CD19-ve cell lines. In similar fashion, the TCRxHER2 DART revealed potent T-cell mediated killing of a panel of HER2 expressing cell lines, including those expressing low HER2 levels (1+ by HercepTest), but was inactive against HER2-negative lines. Analyses of the mechanism of T-cell mediated killing by the DART molecules revealed a dependence on the granzyme B/perforin pathways, with potent killing observed during short-term incubation of the effector populations with target cells and at low E:T ratios. Furthermore, activation of human T cells by DART molecules proceeded strictly in a target-dependent manner. Finally, analyses in xenograft models reconstituted with human peripheral blood mononuclear cells, demonstrated efficient tumor regression by TCR-based DART molecules. In conclusion, T-cell-directed DARTs represent a robust and flexible platform to recruit T-cell populations for cancer cell killing and may offer potential application toward tumor eradication in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5629.

Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcγRIII) on effector cells to an Fv specific for mouse or human CD32B (FcγRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.

Abstract C180: A novel bispecific platform for potent redirected killing of B-cell lymphoma

Abstract Despite significant advances in the treatment of B-cell lymphoma, in many cases the disease remains untreatable or displays recurrence after initial response to therapy. The recent clinical success of bi-specific molecules capable of redirecting T-lymphocyte-mediated cytotoxicity (Bi-specific T-cell Engagers, BiTEs) against B-cell lymphoma provides a feasible therapeutic option. We have developed an alternate bi-specific platform of Dual-Affinity Re-Targeting (DART) molecules that is well suited for clinical applications. Compared to BiTEs, which comprise a pair of single-chain Fv domains in a single polypeptide, DARTs contain two separate chains associated into a heterodimeric, covalently linked, ‘diabody’ structure. We show here that DARTs are very stable in vitro and can be built to recruit and retarget different effector cells, including T cells or CD16- (Fc RIII)-positive cells, against B-cell-lineage targets. A CD3xCD19 DART capable of recruiting cytotoxic T cells against CD19-positive targets mediated extremely potent killing of human peripheral blood B lymphocytes or B-cell lymphoma lines in vitro, with an EC50 that compares favorably to that of a BiTE with identical Fv sequences. Redirected killing of Daudi, Raji, or Nalm6 lymphoma lines was observed in the picomolar range using either freshly purified T cells or peripheral blood mononuclear cells (PBMC) as effector populations. In contrast, no killing was observed against a CD19-negative cell line (RPMI-8226) with either resting or stimulated effector populations, indicating a requirement for CD19 engagement for T-cell-mediated killing by the CD3xCD19 DART molecule. Furthermore, a DART constructed with a humanized version of a pan anti-TCR-beta antibody (TCRxCD19 DART) was as efficient against CD19-positive targets as the CD3-based DART. A CD16xCD32B DART was also constructed to redirect CD16-positive NK and/or mononuclear phagocyte effector cells against CD32B-positive B cells, displaying potent killing against Daudi, Raji or RPMI-8226 cells but not against a CD32B-negative cell line (Ramos). Both TCR- and CD16-based DARTs show potent activity in vivo in several mouse models, including autologous B-cell depletion in huCD32B-transgenic mice, tumor reduction in human Raji Burkitt's lymphoma cell xenografts and Raji cell implants in NOD/SCID mice reconstituted with human PBMC. In conclusion, DARTs represent a robust and flexible platform to recruit different cell populations via various cell surface targets for re-directed killing applications against B-cell malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C180.