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Immunotherapy has emerged as an alternative strategy to treat malignancies in addition to conventional radio- and chemotherapy. There has been a plethora of evidence that the immune system is able to control tumor outgrowth and a number of strategies have been put forward to utilize this ability for immunotherapy. However, some of these strategies have not been very efficient and their success has been limited by tumor evasion mechanisms. A promising approach to engage effector cells of the immune system overcoming some of the escape mechanisms has been introduced more than two decades ago. This approach is based on bispecific antibodies. Here we summarize the evolution of bispecific antibodies, their improvement, remaining obstacles and some controversial reports.

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Redirection of T cells with a first fully humanized bispecific CD33–CD3 antibody efficiently eliminates AML blasts without harming hematopoietic stem cells

The nuclear autoantigen La can be detected on the surface of dying cells. Here we present an assay which enables us to show that La protein is not limited to the surface of dying cells but will be released upon stress-induced cell death. As released La protein tightly binds to the surface of neighboring intact cells we asked the question whether or not La protein could serve as a stress-inducible target e.g. for redirecting of regulatory T cells (Tregs) into damaged tissues to downregulate an immune response. In order to provide first proof of concept we developed a novel fully humanized single-chain bispecific antibody (bsAb) which on the one hand is directed to the La antigen and on the other hand to the CD3 complex of T cells. A cross-linkage of Tregs with La-decorated target cells mediated by this bsAb resulted indeed in the activation of the Tregs in a target-dependent manner. Moreover, such bsAb activated Tregs displayed a potent suppressive capacity and negatively influenced proliferation, expansion and cytokine production of autologous CD4+ and CD8+ Teff cells.

For some instances, protein gels need to be dried after SDS-PAGE, for example, if autoradiography should be performed from radioactive-labeled proteins after their separation on SDS-polyacrylamide gels. Another reason may be to simply store the gel in the laboratory book. Aside from expensive commercial solutions, especially for storage of the dried gel in the lab book, the simple and cheap drying protocol here presented may be sufficient.

For some instances, protein gels need to be dried after SDS-PAGE, for example, if autoradiography should be performed from radioactive-labeled proteins after their separation on SDS-polyacrylamide gels. Another reason may be to simply store the gel in the laboratory book. Aside from expensive commercial solutions, especially for storage of the dried gel in the lab book, the simple and cheap drying protocol here presented may be sufficient.

Minute amounts of proteins are required for immunization of mice for the development of antibodies including monoclonal antibodies. Here, we describe a rapid procedure for the isolation of proteins from polyacrylamide gels after sodium dodecyl sulfate polyacrylamide gel electrophoresis in sufficient amounts for immunization of animals.

Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8+ and CD4+ effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8+ and CD4+ effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4+ T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection.

Despite many years of research and great advances in the field, acute myeloid leukemia (AML) still remains one of the most challenging battle fields in the context of hematologic malignancies treatment. Although AML patients initially respond to conventional chemotherapy, a complete remission is rarely achieved and 5-year survival rates remain low especially in elderly patients. Hence, there is a pressing need for novel effective strategies for AML treatment to prevent relapse and treat minimal residual disease (MRD). The use of recombinant bispecific antibodies (bsAbs) for retargeting effector T lymphocytes towards cancer cells is recently emerging as a promising immunotherapeutic approach for tumor treatment. This class of small molecules is designed to bind simultaneously to a pre-defined tumor-associated antigen (TAA) on tumor cells and the activating CD3 complex on T cells. The cross-linkage of immune effector cell and tumor cell leads to a tumor-specific T cell activation and efficient target cell killing independently of the T cell receptor specificity. However, due to their low molecular mass, bsAbs have a short life span in vivo and consequently have to be continuously administrated to patients over prolonged time spans of several weeks to achieve clinical responses. As an alternative to continuous exogenous infusions of short-lived Abs we examined the use of engineered bone marrow-derived human mesenchymal stem cells (hMSCs) as cellular vehicles for the constant production and secretion of a fully humanized anti-CD33-anti-CD3 bsAb that targets the surface molecule CD33, which is widely overexpressed on AML blasts. Our studies demonstrate that gene-modified hMSCs are effective in releasing the bsAb at sufficient amounts to activate and redirect both human primary CD4+ and CD8+T cells from healthy donors against AML cells expressing varying levels of the CD33 antigen, leading to an efficient T cell-mediated tumor cell killing at low effector to target cell ratios and Ab concentrations. Most importantly, we could demonstrate that patient-derived T cells were able to suppress autologous AML blasts upon Ab-mediated cross-linkage over prolonged period of time without being affected by the presence of the modified hMSCs. Additional improvement of this system was achieved by the artificial expression of T cell co-stimulatory 4-1BB ligand (CD137L) on the hMSCs surface. The additional co-stimulatory signal provided by the engineered hMSCs resulted in an enhanced T cell proliferation, a higher pro-inflammatory cytokine release, and consequently in a more pronounced specific tumor cell killing already at earlier time-points. Taken together, our data could demonstrate that continuous in situ delivery of the anti-CD33-anti-CD3 bsAb by genetically modified hMSCs facilitates efficient activation of T cells for specific and efficient killing of AML blasts over prolonged period of time. Furthermore, as promising perspective of this approach for future in vivo application we are currently investigating on the development of biocompatible synthetic scaffolds as transplantable biomaterial-based production platforms for genetically engineered hMSCs as locally confined vehicle of immunotherapeutics. The implantation of these small engineered devices would ensure that the delivery of the anti-cancer agents can be controlled and stopped after tumor clearance by removing the scaffold at a desired time point. In this way, administration of ex vivo gene-modified hMSCs embedded in appropriate scaffolds would result in a continuous in situ production of recombinant Abs for effective and persistent levels of these therapeutic agents over time with low risk of side effects. Disclosures Cartellieri: Cellex Patient Treatment GmbH, Dresden, Germany: Employment. Ehninger:GEMoaB Monoclonals GmbH, Dresden, Germany: Employment, Patents & Royalties. Ehninger:GEMoaB Monoclonals GmbH, Dresden, Germany: Consultancy, Patents & Royalties. Bachmann:GEMoaB Monoclonals GmbH, Dresden, Germany: Consultancy, Patents & Royalties.

Based on compelling evidence from a vast number of in vitro and in vivostudies, Tregs have become an attractive cell population to treat or even prevent auto- and alloimmunity including Graft-versus-Host disease (GvHD). However, several safety concerns still exist as for example the risk of global immunosuppression using polyclonal Tregs. In fact, experiments in mice showed that adoptive transfer or induction of antigen-specific Tregs is more potent regarding suppression of pathogenic immune responses when compared to polyclonal Treg populations. Unfortunately, the isolation and expansion of naturally occurring antigen-specific Tregs is technically difficult, labour-intensive, and time-consuming. An attractive way to overcome these limitations and to endow polyclonal Treg populations with a desired antigen-specificity is their engraftment with chimeric antigen receptors (CARs). In this context, CAR-modification represents a promising approach to redirect polyclonal Tregs in an antigen-specific manner to suppress ongoing self-destructive immune responses at the site of inflammation. Nevertheless, until now redirection of CAR-engineered T cells is limited to a single target antigen, restricting this approach to an unflexible monospecific therapy. Therefore, we developed a more flexible universal CAR (UCAR) platform that allows redirection of T cells to an in principal unrestricted number of surface antigens. T cells are engrafted with UCARs that bind to a small peptide epitope derived from a human nuclear protein. Cross-linkage to target cells is mediated by independent target modules that provide antigen-specificity and comprise the peptide epitope recognized by the UCAR. In order to target different tissue antigens, the target modules can easily be exchanged. Thereby, once established, the treatment strategy can easily be applied to various auto- and alloimmune diseases. At present, the CD45RA+ population is the Treg subset of choice for a clinical application as these cells have the highest capacity to maintain phenotypic and functional Treg properties upon prolonged ex vivo expansion. Here we show that highly pure, sorted CD4+CD25+CD127lowCD45RA+ Tregs can be genetically manipulated using lentiviral gene transfer, resulting in approximately 70 % of UCAR-expressing Treg cells. The transduction procedure itself did not affect the phenotype of UCAR-engineered Tregs as it was similar to non-transduced wildtype cells. Both Treg populations presevered FOXP3 expression even after prolonged in vitro cultivation (> 95 % FOXP3+). Upon incubation with antigen-positive target cells and a respective target module UCAR-engineered Tregs upregulate the activation markers CD69 and LAP demonstrating that the cells can be restimulated antigen-specifically. Most importantly, UCAR-engrafted Tregs were functionally activated upon antigen encounter, demonstrated by suppression of proliferation and expansion of cocultured autologous T effector cells. Taken together, our results pave the way towards an application of UCAR technology for a site-specific recruitment of CAR-modified Tregs into inflamed tissues aiming at re-establishing immune homeostasis. Due to its high flexibility UCAR-engrafted Tregs can easily and universally be used for treatment of various autoimmune diseases or GvHD just by exchanging the tissue-specific target modules. Disclosures Cartellieri: Cellex Patient Treatment GmbH: Employment. Ehninger:GEMoaB GmbH: Employment, Patents & Royalties. Ehninger:GEMoaB GmbH: Consultancy, Patents & Royalties. Bachmann:GEMoaB GmbH: Consultancy, Patents & Royalties.

We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.

Regulatory T cells (Tregs) play an inevitable role in immune homeostasis by maintaining self-tolerance as well as regulating the magnitude of immune responses against foreign antigens. Over the last few years, the enormous potential of adoptive Treg transfer for treatment of auto- and alloimmunity including Graft-versus-Host disease (GvHD) has been validated in a vast number of in vitro and in vivo studies. For their clinical application, all modes of action should be well understood. Regarding their cytotoxic potential, only few and conflicting data exist. On the one hand, it is assumed that Tregs are capable of inducing apoptosis of effector T cells (Teff) utilizing granzyme/perforin or FasL expression. Others claim that Tregs are not capable of suppressing Teff via programmed cell death pathways but rather induce apoptosis by cytokine deprivation. However, it is of importance to clarify whether Tregs possess a cytotoxic potential particularly when activating the cells antigen-specifically using bispecific antibodies (bsAb). In recent years, bsAb have emerged as promising tools for an antigen-specific immunotherapy of malignant diseases. Their tremendous potential for tumor therapy has been verified in a plethora of in vitro and in vivo studies as well as in first clinical trials. So far, our group was able to demonstrate that not only Teff but also Tregs can be redirected by CD3-engaging bsAb (Koristka et al., J Immunol. 2012; J Autoimmun. 2013). According to a recent presentation (Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research, 2012, abstract nr 4841), bsAb-redirected Tregs can act as killer cells and efficiently mediate cancer cell lysis. In order to shed light onto this controversial issue, we decided to analyze this question in more detail. According to our investigations tumor cell elimination of bsAb-engaged Tregs is largely dependent on the purity of isolated Treg fractions. Tregs isolated on the basis of CD25 expression exhibited a remarkable killing capacity which is most probably due to contaminating CD25+FOXP3- Teff, as highly pure (> 99 %), FACS-isolated CD4+CD25+CD127low Tregs did not display any considerable cytotoxic effect upon cross-linkage to tumor cells via bsAb. The same applies for CD45RA-sorted, expanded Tregs. In comparison to autologous, expanded Teff, tumor cell lysis was negligible. Moreover, the lack of cytotoxicity was independent of the chosen target antigen, as redirecting Tregs with two different bsAb did not result in tumor cell eradication. Besides, upon polyclonal stimulation with conventional aCD3/CD28-coated beads Tregs were not capable of eliminating target cells. Furthermore, as opposed to autologous Teff, Tregs showed only a marginal upregulation of the degranulation marker CD107a when being activated either antigen-specifically via bsAb or polyclonally via beads. Taken together, our findings clearly demonstrate that Tregs bear no considerable cytotoxic potential and hence do not contribute to cancer cell lysis, as recently claimed. On the other hand, the results show that Tregs can be activated by bsAb without the risk of cytotoxic effects against the recognized target cells. This provides the basis for the application of bsAb for a site-specific recruitment of Tregs aiming at attenuating Teff-mediated proinflammatory immune responses and tissue destruction in order to treat auto- and alloimmune diseases including GvHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 1528 Acute myeloid leukemia (AML) is the most common diagnosed cancer affecting the myeloid line of immune cells in adults. Although chemotherapy initially leads to a remission of the disease in 60–80% of the cases, an enormous number of these patients (50–80%) relapse. Hence, new adjuvant therapeutic strategies for the elimination of minimal residual disease (MRD) are needed. One novel attractive approach is based on recombinant bispecific antibodies (bsAb). BsAb are able to mediate a direct cross-link between T cells and tumor cells. This in turn leads to a polyclonal, MHC- and TCR-independent activation of CD4+ and CD8+ T cells for the effective killing of the recognized target cells. A front runner in this field is the bsAb blinatumomab with dual specificity for CD3 and CD19 which is already used successfully in first clinical trials for the treatment of B-cell lymphomas. The development of a novel bsAb is usually very time-consuming and includes a series of individual optimization steps to achieve optimal reactivity with lowest possible side effects. The aim of the present work was therefore to establish a novel, more flexible, less time-consuming, modular cell targeting system and to compare its efficiency and sensitivity with classical bsAb. For the immunotargeting of AML by recombinant antibodies we selected the transmembrane glycoprotein CD33, because 90% of the AML patients have CD33-positive blasts irrespective of the underlying AML-subtype. We developed (i) a novel fully humanized bsAb with dual specificity for CD3 and CD33 and in addition (ii) a novel modular cell targeting system which consists of two different recombinant antibody components (figure 1). The first component (targeting module) is an exchangeable linker module composed of an anti-CD33 single-chain fragment variable (scFv) and a peptide epitope (E). The second component (universal effector module) is a bsAb with antigen binding specificity for the CD3-complex on T cells and for the peptide epitope of the scFv-based linker module. Together both molecules are able to form protein complexes similar to bsAb. Figure 1: Schematic representation of (i) the direct cross-linking system and (ii) the modular cell targeting system Figure 1:. Schematic representation of (i) the direct cross-linking system and (ii) the modular cell targeting system The functionality of both immunotargeting systems was examined by performing chromium (51Cr) release assays. As target cells both AML blasts from patients and artificially overexpressing CD33-positive tumor cells were used. Both the direct targeting system consisting of a classical novel humanized anti-CD33-anti-CD3 bsAb and the modular cell targeting system efficiently killed CD33-positive targets cells down to picomolar concentrations (figure 2). Figure 2: Tumor cell lysis mediated by classical bsAb and the modular cell targeting system was determined by performing a chromium release assay. For this purpose, CD33-positive, 51Cr loaded target cells were incubated with PBMCs as effector cells. Co-cultivation occurred either in the presence or absence of recombinant Ab by using an effector to target cell ratio of 10: 1 and different amounts of recombinant Abs. Figure 2:. Tumor cell lysis mediated by classical bsAb and the modular cell targeting system was determined by performing a chromium release assay. For this purpose, CD33-positive, 51Cr loaded target cells were incubated with PBMCs as effector cells. Co-cultivation occurred either in the presence or absence of recombinant Ab by using an effector to target cell ratio of 10: 1 and different amounts of recombinant Abs. Compared to the classical bsAb the novel modular cell targeting has a series of advantages: Just by replacing the targeting module the system can be easily modified for retargeting of T cells against other tumor antigens. Moreover, using linker modules with dual specificities enables a multi-specific targeting which may improve the targeting specificity and thereby might increase the success of an immunotherapy. Taken together, here we present for the first time two novel fully functional humanized, recombinant Ab-based systems for the immunotargeting of CD33+ AML blasts by CD3+ T cells. The obtained data indicate that both the direct cross-linking anti-CD3-anti-CD33 bsAb and the modular cell targeting system hold great potential as an adjuvant cancer immunotherapy for the elimination of MRD in AML-patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4041 Hematopoietic stem cell transplantation is a commonly used treatment for various hematological malignancies. However, a life-threatening complication following this therapy is the development of Graft versus Host Disease (GvHD), during which transplanted donor immune cells attack the host and lead to severe inflammatory responses and tissue damage. Given the key role of regulatory T cells (Tregs) in immune homeostasis and peripheral tolerance, the adoptive transfer of these cells may represent a promising therapeutic opportunity for the treatment of GvHD. This approach has been proven successful to enhance graft acceptance and prevent experimental GvHD in several animal models. However, it becomes increasingly evident that antigen-specific Tregs are more efficient than polyclonal Treg populations. The antigen-specificity enables the cells to exert their suppressive effect locally at the appropriate sites of inflammation. Furthermore, the application of antigen-specific Tregs might lower the risk of unfavourable systemic immunosuppression. Nevertheless, one of the main obstacles for their clinical use is the isolation and expansion of therapeutically relevant numbers of antigen-specific Tregs. In light of these arguments, bispecific antibodies (bsAb) could provide a promising tool for a target-dependent tissue specific redirection of polyclonal Tregs. BsAb redirect T cells to target cells by cross-linking their activating CD3 receptor and any chosen antigen on the surface of the target cell. Several studies have proven that CD8+ and CD4+ effector T cells can be successfully activated by bsAb both in vitro and in vivo. However, so far nobody has ever investigated whether Tregs can be redirected with bsAb. Using bsAb against CD3 and two different target antigens we first examined the expression of different activation associated markers on CD4+CD25+ Tregs. We could show that incubation of Tregs with a bsAb in the presence of the respective target antigen leads to the upregulation of CD25, CD69 and LAP (figure 1). Furthermore, we analyzed the cytokine production profile of the bsAb-activated Tregs. Culturing the cells together with target cells and a cross-linking bsAb triggers the release of the immunomodulatory cytokine IL-10. One prerequisite for the treatment of graft rejection and GvHD with bsAb-redirected Tregs is their suppressive efficacy upon antigen-specific activation via bsAb. Therefore we monitored the ability of bsAb-redirected Tregs to inhibit effector mechanisms of autologous T helper cells. Corresponding experiments could demonstrate the striking capacity of redirected Tregs to suppress proliferation, CD25 upregulation and cytokine production of co-cultured effector T cells (figure 2).Figure 1:Expression of different activation associated markers on Tregs incubated without (−) or with a bsAb (+) and the respective target antigen.Figure 1:. Expression of different activation associated markers on Tregs incubated without (−) or with a bsAb (+) and the respective target antigen.Figure 2:A) CFSE-labeled effector T cells (Teff) were cultured together with either unlabeled autologous effector T cells or Tregs at effector:suppressor ratios of 1:1 or 4:1 in the presence (+) or absence (−) of a bsAb and the respective target antigen. B) Cytokine secretion of bsAb-activated Teff in the presence (white bars) or absence (black bars) of autologous bsAb-activated Tregs.Figure 2:. A) CFSE-labeled effector T cells (Teff) were cultured together with either unlabeled autologous effector T cells or Tregs at effector:suppressor ratios of 1:1 or 4:1 in the presence (+) or absence (−) of a bsAb and the respective target antigen. B) Cytokine secretion of bsAb-activated Teff in the presence (white bars) or absence (black bars) of autologous bsAb-activated Tregs. Taken together, we give evidence for the first time that bsAb can redirect Tregs against a surface antigen independently of their T cell receptor specificity. In view of these results, an antigen- and/or site-specific retargeting of Tregs using bsAb may open novel therapeutic approaches for a long-term establishment of tolerance against allogenic transplants and therefore would offer a new treatment option for severe GvHD after allogenic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3–anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.

Abstract 2618 Adoptive transfer of antigen-specific T cells emerged as an attractive strategy to provide cancer patients with immune cells of a desired specificity. The efficacy of such adoptive transfers has been demonstrated in clinical studies. However, enrichment and expansion of tumor-specific T cells are time-consuming and often ineffective, due to the low frequency of tumor-specific precursors in vivo. Alternatively, polyclonal T cells can be genetically modified with chimeric antigen receptors (CARs) to provide these cells with new antigen specificities. CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen fused to a signaling chain derived from a lymphocyte activating receptor. The chimeric receptor approach is able to bypass many of the mechanisms by which tumors avoid immunorecognition, such as MHC down-regulation, lack of expression of costimulatory molecules, and induction of T cell suppression. Acute myeloid leukemia (AML) is an intrinsically resistant disease and even though the majority of the patients initially respond to chemotherapy, the 3-year survival rate is still low. A promising target for immunotherapy of AML is CD33, which is absent on normal pluripotent hematopoietic stem cells and normal tissues, but is present on leukemic blasts in 85–90 % of adult and pediatric AML cases independent of the subtype of AML. Novel human CD33-specific CARs were constructed by fusing a CD33 specific scFv in series with CD3ζ chain plus an additional costimulatory sequence derived from CD28 (Fig. 1A). Both native human CD8+ and CD4+ T cells engrafted with CD33-specific CARs exhibited antigen-specific cytokine secretion, proliferation and target cell lysis (Fig. 1B, C). Moreover, AML blast derived from patients were efficiently killed by allogeneic CAR-engrafted T cells (Fig. 1D). Next, the CD33-specific scFv was fully humanized and afterwards incorporated into the CAR constructs. With this humanized CAR engrafted T cells from AML patients could be redirected against CD33+ cell lines and autologous AML blasts. Upon antigen-recognition, the modified T cells secreted high amount of inflammatory cytokines and efficiently killed the target cells.Fig. 1:Human CAR-engrafted T cells mediate effector functions against CD33+ target cells.A. Schematic representation of the CD33-specific CARs. VL: variable light chain; VH: variable heavy chain; E-Tag: linker epitope in between the VL and the VH chain.Fig. 1:. Human CAR-engrafted T cells mediate effector functions against CD33+ target cells. / A. Schematic representation of the CD33-specific CARs. VL: variable light chain; VH: variable heavy chain; E-Tag: linker epitope in between the VL and the VH chain.B. Cytotoxic effector functions of CAR engrafted human CD8+ and CD4+ T cells against the CD33+ blast line U937 were measured in a standard chromium release assay after 6h of co-incubation.B. Cytotoxic effector functions of CAR engrafted human CD8+ and CD4+ T cells against the CD33+ blast line U937 were measured in a standard chromium release assay after 6h of co-incubation.C. Killing of patient-derived AML blast by allogeneic CAR engrafted T cells was measured in a flow cytometer by exact cell count after 48h of co-cultivation. Three independent donor/patient pairings are shown.C. Killing of patient-derived AML blast by allogeneic CAR engrafted T cells was measured in a flow cytometer by exact cell count after 48h of co-cultivation. Three independent donor/patient pairings are shown. Until now, one major obstacle for an adoptive therapy of genetically modified T cells is the limited amount of T cells that can be isolated from AML patients and modified in vitro. For an efficient in vitro expansion restricted to CAR modified T cells from patients we developed a new method based on a novel CAR-mediated strategy. For this purpose, magnetic beads were coated with an antibody recognizing an epitope (Fig. 1A, E-Tag) which we included in the linker domain between the heavy and the light chain of the scFv portion of the CAR. Adding such magnetic beads to cultures of CAR modified T cells, the CAR engrafted T cells were expanded to similar extends as polyclonal T cell populations with anti-CD3/anti-CD28 coated beads. Furthermore, the antibody-coated beads can be used to isolate the CAR engrafted T cells after expansion and get rid of any contaminating non-modified cells. It may also be useful for elimination of CAR expressing T cells in vivo if necessary. The feasibility of the described method was not limited to CD33 specific CARs but was also functional for CARs equipped with scFvs of other specificities. Therefore, it might be universally applicable for the expansion and preparation of CAR modified T cell grafts in vitro before adoptive transfer back in patients. Taken together, we describe novel humanized CD33-specific CARs that (I) can be specifically expanded, (II) specifically eliminated, if necessary, and (III) may therefore become a novel potent treatment option for a cellular therapy of AML patients. Disclosures: No relevant conflicts of interest to declare.

New promising candidates for cancer immunotherapy are bispecific antibodies (bsAbs) which have already shown a convincing anti-tumor effect in first clinical trials. BsAbs are composed of two single-chain fragments variable (scFvs), derived from the variable heavy and light chain of a monoclonal Ab (mAb), which bind to the activating CD3-complex on T cells and a tumor-associated antigen (TAA) on cancer cells. Consequently, the cross-linkage of effector and target cells by bsAbs results in an efficient T cell-mediated cancer cell killing. However, several serious adverse effects were observed in patients after application of Ab constructs. Most critical is an overall activation of T cells which bears the risk of a systemic release of pro-inflammatory cytokines. With respect to the idea that the CD3 binding of an Ab construct is critical for T cell activation, the CD3 domain has to be optimized in order to prevent or even reduce unspecific T cell responses. For this reason, we cloned several anti-CD3 scFvs derived from different anti-CD3 mAb clones and compared their functionality and safety. The anti-CD3 scFv with lowest risk of side effects was introduced in different anti-TAA bsAbs. Functional and safety studies in vitro revealed that development of each bsAb requires extensive and time-consuming optimization steps to gain high efficacy but low risk of side effects. To improve the process of Ab engineering, we recently introduced a novel modular Ab platform that can be rapidly and cost-effectively adapted for redirection of T cells to any TAA (Arndt and Feldmann et al. accepted for publication in Leukemia 2013). In this novel modular system the dual specificity of a conventional bsAb is distributed to two separate Ab modules, (i) the effector module and (ii) the target module. Only the complex of both Ab modules mediates the cross-linkage of effector and target cells resulting in T cell activation and redirected cancer cell lysis similar to conventional T cell engaging bsAbs. The universal effector module is a well optimized bsAb balancing efficacy and safety. It binds CD3 on T cells and the E5B9 tag of the target module. The individual target module comprises an anti-TAA scFv and the peptide epitope E5B9. For treatment of lymphoid or myeloid malignancies a series of different target modules were designed. In vitro and in vivo data clearly underline that the combination of the established effector module with different target modules efficiently activated T cells against hematological malignancies. Both CD4+ and CD8+ T cells from healthy donors were efficiently activated to kill TAA-positive tumor cell lines at low effector to target cell ratios and Ab concentrations in a tumor-specific manner. Most importantly, we could demonstrate that patient-derived T cells were able to kill autologous malignant cells upon Ab-mediated cross-linkage. Another unique feature of the modular system is that multispecific or multifunctional target modules could be easily included to further improve the therapeutic effect. In order to increase anti-tumor specificity and reduce the risk of tumor escape variants, bispecific target modules that recognize different TAAs at the same time, were constructed. Cytotoxicity assays investigating dual targeting of double-positive tumor cells via the modular system demonstrated that target cell lysis can be considerably improved in comparison to single targeting modules. Moreover, proliferation and cytokine release of redirected T cells could be enhanced by supplying costimulatory immunoligands (e.g. 4-1BBL and Ox40L) via target modules. In summary, we developed a multifunctional and highly flexible modular system based on an anti-CD3 domain with lowest risk of side effects. In contrast to conventional anti-TAA-anti-CD3 bsAbs, the development of a novel modular system for different clinical indications is much easier and less time-consuming. Once the effector module containing the critical CD3 domain is optimized, the modular system can be flexibly applied to target any TAA simply by replacing the target module. Application of bispecific target modules or providing costimulatory signals via the modular system might prolong immune responses and further increase anti-tumor specificity and activity. Finally, the novel modular platform represents a valuable and promising tool for cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.