CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell therapy can be associated with significant toxicities. CAR-engineered natural killer (NK) cells provide a safer alternative while maintaining anti-tumor effects. Activated NK (aNK) cells are a clinical-grade cellular product obtained from the NK-92 cell line that have demonstrated both safety and potent cytotoxicity toward a wide range of cancers in phase 1 trials. Genetically engineered variants of aNK cells expressing a high-affinity Fc receptor (haNK) or co-expressing a CAR (t-haNK) are currently in phase 1/2 clinical trials. A key factor in the efficacy of cellular immunotherapies is biodistribution and tumor infiltration, which affect the local effector:target ratio. The chemokines CCL19 and CCL21 can drive recruitment of CCR7 receptor-expressing immune cells to secondary lymphoid organs. METHODS: Since NK-92 cells do not spontaneously express CCR7, clinical-grade aNK cells were transfected with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR and a high-affinity CD16 Fc receptor. RESULTS: CCR7-engineered CD19 t-haNK showed significant migration in vitro toward K562 cells engineered to secrete CCL19. This observation was confirmed in a NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model in which subcutaneous tumors of CCL19-expressing K562 cells displayed a higher number of infiltrating CCR7_CD19 t-haNK cells than CCR7-negative CD19 t-haNK cells. In NSG mice inoculated either intravenously or subcutaneously with CCL19-secreting Raji cells, treatment with CCR7_CD19 t-haNK improved survival and tumor control compared with CD19 t-haNK or vehicle. CONCLUSIONS: Expression of CCR7 receptor by off-the-shelf t-haNK cells improves their homing toward lymph node chemokines both in vitro and in vivo, resulting in superior tumor control.

Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting.

BACKGROUND: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors. METHODS: Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. RESULTS: Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals. CONCLUSIONS: These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.

An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

Natural Killer Cells for Immunotherapy - Advantages of the NK-92 Cell Line over Blood NK Cells.

Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity.

Multiple natural killer (NK) cell-based anticancer therapies are currently under development. Here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (CARs) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγRIII). First, we compared in vitro the abilities of NK-92 cells expressing CD20-targeting CARs to kill primary chronic lymphocytic leukemia (CLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγRIII combined with either of the anti-CD20 monoclonal antibodies (mAbs) rituximab or ofatumumab. We found that CAR-expressing NK-92 cells effectively kill NK cell-resistant primary CLL cells and that such a cytotoxic response is significantly stronger than that resulting from ADCC. For studying CAR-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human BCR-ABL1+ cell lines SUP-B15 (CD19+CD20-) and TMD-5 (CD19+CD20+), two acute lymphoblastic leukemia (ALL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing CD19-targeting CARs eliminated SUP-B15 cells, whereas they had no such effect on TMD-5 cells. However, the intrafemoral injection of NK-92 cells expressing CD19-targeting CAR resulted in the depletion of TMD-5 cells from the bone marrow environment. Comparative studies in which NK-92 cells expressing either CD19- or CD20-targeting CARs were directly injected into subcutaneous CD19+CD20+ Daudi lymphoma xenografts revealed that CD20-targeting CAR is superior to its CD19-specific counterpart in controlling local tumor growth. In summary, we show here that CAR-expressing NK-92 cells can be functionally superior to ADCC (as mediated by anti-CD20 mAbs) in the elimination of primary CLL cells. Moreover, we provide data demonstrating that the systemic administration of CAR-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of CAR-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma.

Identification and characterization of the RLIP/RALBP1 interacting protein Xreps1 in Xenopus laevis early development.

BACKGROUND: The FGF/Ras/Ral/RLIP pathway is required for the gastrulation process during the early development of vertebrates. The Ral Interacting Protein (RLIP also known as RalBP1) interacts with GTP-bound Ral proteins. RLIP/RalBP1 is a modular protein capable of participating in many cellular functions. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of RLIP in early development, a two-hybrid screening using a library of maternal cDNAs of the amphibian Xenopus laevis was performed. Xreps1 was isolated as a partner of RLIP/RalBP1 and its function was studied. The mutual interacting domains of Xreps1 and Xenopus RLIP (XRLIP) were identified. Xreps1 expressed in vivo, or synthesized in vitro, interacts with in vitro expressed XRLIP. Interestingly, targeting of Xreps1 or the Xreps1-binding domain of XRLIP (XRLIP(469-636)) to the plasma membrane through their fusion to the CAAX sequence induces a hyperpigmentation phenotype of the embryo. This hyperpigmented phenotype induced by XRLIP(469-636)-CAAX can be rescued by co-expression of a deletion mutant of Xreps1 restricted to the RLIP-binding domain (Xreps1(RLIP-BD)) but not by co-expression of a cDNA coding for a longer form of Xreps1. CONCLUSION/SIGNIFICANCE: We demonstrate here that RLIP/RalBP1, an effector of Ral involved in receptor-mediated endocytosis and in the regulation of actin dynamics during embryonic development, also interacts with Reps1. Although these two proteins are present early during embryonic development, they are active only at the end of gastrulation. Our results suggest that the interaction between RLIP and Reps1 is negatively controlled during the cleavage stage of development, which is characterized by rapid mitosis. Later in development, Reps1 is required for the normal function of the ectodermic cell, and its targeting into the plasma membrane affects the stability of the ectoderm.

Comparison of mRNA and lentiviral based transfection of natural killer cells with chimeric antigen receptors recognizing lymphoid antigens.

Natural killer (NK) cells can be engineered to kill resistant B-lymphoid cell lines and primary B-cell chronic lymphocytic leukemia (B-CLL) cells after transfection with chimeric antigen receptors (CARs) recognizing CD19 or CD20. Here we compared mRNA electroporation with lentiviral vector (LV) transduction for both CARs. Transfection efficiency and cytotoxicity of previously NK-92 resistant CLL cells were significantly higher after mRNA electroporation than after LV transduction. Further cell sorting of LV-transduced NK-92 cells resulted in a highly enriched population of transduced cells with significant target cell lysis. Compared to NK-92 cells, peripheral blood and cord blood cells consistently showed < 10% transfection efficiency with mRNA, while LV transduction varied between 8 and 16% for peripheral blood and 12 and 73% for cord blood. These results suggest that LV should be used to achieve sufficient transgene expression if blood NK cells are considered for CAR transduction. Transfection with mRNA results in clinically relevant levels of transfection only in NK-92 cells.

Variable contribution of monoclonal antibodies to ADCC in patients with chronic lymphocytic leukemia.

Monoclonal antibodies (mAbs) are increasingly used in treatment protocols for chronic lymphocytic leukemia (CLL). Here we determined (i) the extent of antibody-dependent cellular cytotoxicity (ADCC) of four different mAbs against primary CLL cells, (ii) whether ADCC correlates with antigen density on CLL cells, and (iii) whether allogeneic natural killer (NK) cells display superior ADCC than autologous. Effector cells for ADCC were (i) NK-92 cells not expressing FcR, (ii) NK-92 cells transfected with a high-affinity Fc receptor, (iii) autologous NK cells from patients with CLL, (iv) allogeneic NK cells. Results suggest that ADCC contributes to killing of CLL cells by anti-CD20 antibodies (rituximab and veltuzumab), whereas mAbs against CD22 (epratuzumab) and CD23 (lumiliximab) showed minimal ADCC. The magnitude of anti-CD20 mediated ADCC did not correlate with antigen density of CD20. ADCC was not influenced by the FcR genotype expressed by autologous NK cells. Allogeneic NK cells were superior to autologous NK cells in killing primary CLL cells.

Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells.

An emerging treatment option for chronic lymphocytic leukemia (CLL) is to make cytotoxic immune cells express a chimeric antigen receptor (CAR) that recognizes specific surface molecules on CLL cells. Here an mRNA coding for an anti-CD19 CAR was transfected into the NK-92 cell line by electroporation. In contrast to cDNA, mRNA resulted in high transfection efficiency (47.2 +/- 8% versus <5% for cDNA) with minimal effect on cell viability. NK-92 cells expressing anti-CD19 CAR killed previously resistant CD19+ B-ALL cell lines, as well as primary CLL cells and therefore may present a safe, cell-based, targeted treatment for patients with CLL.

Umbilical cord mesenchymal stem cells increase expansion of cord blood natural killer cells.

Natural killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cell progenitors with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence, it is attractive to expand and potentially utilize these cells for adoptive immunotherapy. In this study, CB mononuclear cells were CD3-depleted by immunomagnetic microbead selection to remove T cells. This CD3(dep) CB-MNC fraction was then plated for ex vivo expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal stem cells (UC-MSC), with or without cytokines that have been shown to be critical for NK expansion: IL-2, IL-15, IL-3, and FLT-3L. At an average of 2 weeks of culture, there was significantly higher expansion (64.7 +/- 8.4-fold) of CD56(+)/CD3(-) NK cells in the presence of the UC-MSC feeder layer and cytokines compared to controls (no increase with feeder layer only and 6.4 +/- 1.5-fold increase with cytokines only, P < .05). Contact between CD3(dep) CB-MNC cells and UC-MSC augmented NK expansion. The combination of all 4 cytokines was superior to IL-2 alone or 2 cytokines combinations: mean 64.7 +/- 8.4-fold expansion with 4 cytokines combination versus IL-2 alone, IL-2 + FLT-3L, IL-2 + IL-15 or IL-2 + IL-3 (12.2 +/- 2.0, 14.4 +/- 2.4, 10.4 +/- 4.1, 25.2 +/- 8.1 respectively). We also observed that only fresh CD3(dep) CB-MNC preparations could be expanded reliably, whereas frozen and thawed CD3(dep) CB-MNC cells did not expand consistently (mean fold increase 6.5 +/- 3.2). Cytotoxicity of expanded NK cells was compared with NK cells from fresh and overnight IL-2 activated CD3(dep) CB-MNC. Whereas fresh cells displayed no discernible killing, strong cytotoxicity against K562, Raji, REH, and SUP-B15 cells lines was noted after overnight activation in IL-2. Cytotoxicity of expanded NK cells against Raji, REH, and SUP-B15 was lower, which, however, correlated with a predominant expansion of CD56(+)/CD16(-) cells known to have less cytolytic activity than CD56(+)/CD16(+). To test the transfection efficiency in NK cells, fresh or expanded CD3(dep) CB-MNC cells were electroporated with either DNA or mRNA constructs for GFP. DNA had a low transfection efficiency (<10%), whereas the one for mRNA reached 52%, but at the cost of significant cell death. Our results suggest that CB NK cell progenitors can be expanded to obtain large numbers by using an irradiated feeder of UC-MSC. They maintain an elevated cytotoxic profile, and may be genetically manipulated-all characteristics that make them suitable for cellular therapies.

Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation.

The Wharton's jelly of the umbilical cord is rich in mesenchymal stem cells (UC-MSCs) that fulfill the criteria for MSCs. Here we describe a novel, simple method of obtaining and cryopreserving UC-MSCs by extracting the Wharton's jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum. This direct freezing of cord microparticles without previous culture expansion allows the processing and freezing of umbilical cord blood (UCB) and UC-MSCs from the same individual on the same day on arrival in the laboratory. UC-MSCs produce significant concentrations of hematopoietic growth factors in culture and augment hematopoietic colony formation when co-cultured with UCB mononuclear cells. Mice undergoing transplantation with limited numbers of human UCB cells or CD34(+) selected cells demonstrated augmented engraftment when UC-MSCs were co-transplanted. We also explored whether UC-MSCs could be further manipulated by transfection with plasmid-based vectors. Electroporation was used to introduce cDNA and mRNA constructs for GFP into the UC-MSCs. Transfection efficiency was 31% for cDNA and 90% for mRNA. These data show that UC-MSCs represent a reliable, easily accessible, noncontroversial source of MSCs. They can be prepared and cryopreserved under good manufacturing practices (GMP) conditions and are able to enhance human hematopoietic engraftment in SCID mice. Considering their cytokine production and their ability to be easily transfected with plasmid-based vectors, these cells should have broad applicability in human cell-based therapies.

Recruitment of Cdc42 through the GAP domain of RLIP participates in remodeling of the actin cytoskeleton and is involved in Xenopus gastrulation.

The transduction pathways that branch out of fibroblast growth factor signaling are essential for the induction of the mesoderm and the specification of the vertebrate body plan. One of these pathways is thought to control remodeling of the actin cytoskeleton through the Ral binding protein (RLIP also known as RalBP1), an effector of the small G protein Ral. RLIP contains a region of homology with the GTPase-activating protein (GAP) domain involved in the regulation of GTPases of the Rho family. We demonstrate here that the GAP domain of RLIP is responsible for the stability of the actin cytoskeleton in Xenopus laevis embryos. We also demonstrate that the complete N-terminal domain of RLIP containing the mu2 binding domain (mu2BD) and the GAP domain induces disruption of the actin cytoskeleton when targeted to the plasma membrane. Neither domain, however, has any effect on the actin cytoskeleton when individually targeted to the plasma membrane. We also determined that Cdc42-GDP, but neither Rac-GDP nor Rho-GDP, rescues the effect of expression of the membrane-localized Xenopus RLIP on the actin cytoskeleton. We show that the GAP domain of RLIP interacts in vivo with Cdc42-GTP and Cdc42-GDP. Finally, a single mutation (K244A) in the GAP sequence prevented embryos from gastrulating. These results demonstrate that to participate in the control of the actin cytoskeleton, RLIP needs its complete N-terminal region coding for the mu2BD and the GAP domain. We suggest that RLIP, by coordinating two complementary mechanisms, the endocytosis of clathrin-coated pits and the remodeling of cortical actin, participates in the gastrulation process.

RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development.

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.

Control of embryonic Xenopus morphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway.

Ras proteins mediate biological responses through various effectors and play a key role in relaying the Fibroblast Growth Factor (FGF) mesoderm induction signal during embryogenesis of the frog, Xenopus laevis. One Ras effector pathway involves the activation of the small G protein Ral. In the present study, we have investigated the role of key components in the Ral branch of FGF and Ras signalling during early Xenopus development. Treatment of animal caps with bFGF, which converts prospective ectoderm to mesoderm, activates Xral. The Ras mutant 12V37G, which can bind to Ral-GDS but not Raf, also activates Xral as well as causing developmental defects and cortical F-actin disassembly. A similar phenotype is induced by Ral-GDS itself. FGF-induced expression of several signature mesodermal genes, by contrast, is independent of Xral signalling. This and other data suggest that the RalB branch of Ras and FGF signalling regulates the actin cytoskeleton and morphogenesis in a transcriptionally independent manner. We also find Xral to be specifically activated in the marginal zone of Xenopus embryos, and find that disruption of the Ral pathway in this region prevents closure of the blastopore during gastrulation. We conclude that Ral signalling is autonomously required by mesodermal cells to effect essential morphogenetic changes during Xenopus gastrulation.