Background Combining interleukin-2 (IL-2) agonism with programmed cell death protein 1 (PD-1) checkpoint inhibition has shown synergistic potential in reinvigorating antitumor T cell responses. However, integrating these two mechanisms within a single molecule has been challenging due to competing requirements for PD-1 engagement and IL-2 receptor signaling. ANV600 is a novel bispecific antibody–cytokine fusion protein that targets a non-blocking epitope on PD-1, enabling cis-targeted IL-2Rβγ agonism while preserving combinability with therapeutic PD-1 inhibitors. This design allows for selective expansion of tumor antigen-specific T cells while avoiding the systemic toxicity and regulatory T cell (Treg) expansion associated with conventional IL-2 therapies.Methods The PD-1-targeting antibody used in ANV600 was generated by immunization of humanized mice and selected for its ability to bind PD-1 without blocking the binding epitope of PD-1 checkpoint blocking agents. ANV600 was evaluated in multiple syngeneic tumor models using human PD-1 transgenic mice. Tumor-infiltrating lymphocytes were analyzed to assess the selectivity of ANV600 for PD-1+ T cell subsets. Combination studies with pembrolizumab and nivolumab were performed to assess synergy with checkpoint inhibitors.Results ANV600 significantly inhibited tumor growth as monotherapy across multiple models, including the immune checkpoint-resistant B16F10 melanoma. By targeting PD-1, ANV600 selectively expanded tumor antigen-specific CD8+T cells, particularly progenitor exhausted (Tpex) and cytotoxic exhausted (Tcex) subsets, while sparing Tregs and NK cells. Combination with pembrolizumab and nivolumab resulted in additive effects, consistent with the complementary roles of PD-1 blockade in expanding Tpex cells and IL-2Rβγ signaling in reprogramming Tcex cells. ANV600’s efficacy was dependent on CD8+T cells and primarily driven by tumor-resident T cells, as it remained effective despite blocked lymph node trafficking (FTY720) but was abrogated on CD8+ T cell depletion.Conclusions ANV600 represents a novel approach to delivering IL-2Rβγ agonism specifically to PD-1+ cells while preserving the binding site for PD-1 checkpoint inhibitors. By targeting a non-blocking epitope on PD-1, ANV600 enables the selective expansion of tumor-reactive CD8+ T cells while allowing independent and optimized dosing of both agents. This design ensures combinability with PD-1 inhibitors at clinically relevant doses, including in patients previously treated with checkpoint blockade. These findings support the clinical development of ANV600 as both a monotherapy and a combination therapy in cancer immunotherapy.

Novel engineered IL-2 agonists strive to increase the therapeutic window of aldesleukin (human IL-2) by increasing selectivity toward effector over regulatory T cells and reducing dose-limiting toxicities. Here we describe ANV419, an IL-2/anti-IL2 antibody fusion protein designed for selective IL-2 receptor βγ (IL-2 Rβγ) activation by sterically hindering IL-2 from binding to IL-2 Rα. The fusion protein has an IL-2 connected to the light chain complementarity-determining region (CDR) domain of a humanized antibody that binds to IL-2 at the same epitope as IL-2 Rα. Optimization of the selectivity and pharmacological properties led to the selection of ANV419. ANV419 preferentially expands CD8+ T cells and natural killer (NK) cells over Tregs and can be safely administered at doses that elicit strong pharmacodynamic effects and efficacy in mouse tumor models. Its anti-tumor efficacy was enhanced when combined with programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint inhibitors. ANV419 also enhances the NK cell killing capacity and increases tumor growth inhibition when used alongside trastuzumab in a Her-2+ xenograft mouse model. In cynomolgus monkeys, the estimated half-life of ANV419 is 24 h, and doses that induced sustained expansion of effector cells were well tolerated without the severe toxicities typically observed with high-dose IL-2. These data support the clinical development of ANV419 in solid tumors and hematological malignancies as monotherapy and in combination with checkpoint inhibitors or agents that induce antibody-dependent cellular cytotoxicity. ANV419 is currently in Phase 1/2 clinical development and may provide cancer patients with a wider therapeutic window than aldesleukin.

Background Uveal melanoma (UM) is a poorly immunogenic melanoma subtype with low objective response rate (ORR) to immune checkpoint inhibitors (ICI). With a strong predilection for hepatic metastases, UM patients often receive liver-directed therapies, such as hepatic artery embolization. We hypothesized that transarterial immune-embolization (TAIE) with GM-CSF to treat hepatic metastases may synergize with concurrent use of ICIs, plausibly through improved antigen presentation. Methods This single-center retrospective study includes UM patients with liver-predominant metastatic disease who received TAIE using a combination of GM-CSF (2000 mcg) and lipiodol for up to 10 treatments in an alternating lobar fashion, with/without concurrent systemic ICI (defined as administered within 3 months of starting TAIE). Efficacy endpoints included investigator-assessed ORR per RECIST 1.1, progression-free survival (PFS) and overall survival (OS). Safety endpoints included adverse events (AEs), related to TAIE and/or ICI. Results Between 2016–2023, 18 metastatic UM patients (8M; 10F) with median age 64 (range 46–80) years received 83 IE treatments (median 3, range 1–10). Median follow up was 19.3 (range 1.7 – 47.1) months. Fourteen of 18 (78%) patients received concurrent ICI (n = 10 with combination anti-CTLA-4/PD-1, n = 4 with anti-PD-1). ORR was 17% (3/18), with all 3 patients experiencing partial responses lasting 4.2, 28.1+ and 38.6 months, respectively, while receiving concurrent ICI. Seven (39%) patients had stable disease as best response, resulting in a disease control rate of 56% (10/18). Median OS from first TAIE treatment was 35 (range 1.7- 39.2+) months. Concurrent IE with ICI was generally tolerated well, except one of 13 (8%) patients requiring hospitalization for transient distributive shock (n=1), which resolved with supportive care. Immune-related AE (IRAE) were only observed in patients receiving combination ICI with anti-CTLA-4/PD-1, including hepatitis (n= 5; G2 in 1 and G3 in 4; 4 required steroids and all resolved), pneumonitis (n=1; G1), pancreatitis (n=1, G2), colitis (n=1; G3) and adrenal insufficiency (n=1; G3); four of these seven patients resumed PD-1 monotherapy without further AEs. Conclusions Concurrent administration of liver-directed therapy with GM-CSF TAIE and systemic ICI, including anti-CTLA4/PD-1 combination, is safe and feasible, and can lead to sustained clinical benefit in a subset of UM patients. For this poorly immunogenic cancer with a characteristic predilection for hepatic metastases, liver-directed novel immunotherapy approaches offer a unique opportunity to synergize with systemic immunotherapies. Ethics Approval University of Washington IRB Committee D approved the study: Clinical Outcome of Immunotherapy in Melanoma and Other Skin Cancer Patients, Investigator: Shailender Bhatia, IRB ID: STUDY00011495. IRB determined that consent was waived due to minimal risk.

Non-alpha IL-2-based therapeutic modalities with preferential signaling through the IL-2 beta(CD122)/gamma(CD132) receptor are in clinical development and have the potential to substantially increase the therapeutic index of recombinant IL-2 (aldesleukin) for cancer therapy. ANV600 is a novel bispecific compound, which features an anti-IL-2 antibody/IL-2 fusion protein and a proprietary hPD-1 binding moiety for specific delivery of the non-alpha IL-2 to tumor antigen experienced PD-1+ T cells. Compared to the untargeted control, ANV600 has increased potency to induce STAT5 phosphorylation in human PD-1+CD8 T cells in vitro. At the same time, stimulation with ANV600 results in markedly reduced STAT5 phosphorylation on Treg cells compared to aldesleukin. In activated human PBMCs, ANV600 induces PD-1 co-internalization with the CD122-CD132 complex, reducing detectable PD-1 on CD8 and CD4 T-cells. Surface PD-1 levels decrease is not observed on cells incubated with untargeted bispecific compound. In transgenic human PD-1 mice ANV600 treatment leads to marked tumor growth retardation in the B16F10 and MC38 subcutaneous tumor models compared to untargeted compound and vehicle. Immunophenotyping of the tumor infiltrating lymphocytes revealed a dose-dependent increase of intratumoral stem-like PD-1+T cells and cytotoxic GrzB+PD-1+T cells in mice treated with ANV600. ANV600 has potent and selective effects on antigen-experienced CD8 T cells both in human PBMCs and mouse syngeneic tumor models. This agent may be a promising anti-tumor therapeutic against poorly immunogenic tumors and warrants further pharmaceutical development. Citation Format: Patrizia Murer, Ulisse Salazar, Nicole Egli, Laetitia Petersen, Pia Neubert, Kirsten Richter, Christian Stocker, Alexander Rau, Andreas Katopodis, Christoph Huber. ANV600 is a potent, Cis-signaling, non-alpha IL-2 agonist which efficiently expands intratumoral stem-like CD8 T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4127.

e21552 Background: IL-2R agonists that are well tolerated and can selectively enhance immune activation may improve outcomes of patients with cancer. ANV419 is a potent and highly selective IL-2Rβγ binding agonist, consisting of an antibody specific for the IL-2Rα-binding domain of IL-2, fused to native hIL-2. It is currently being investigated in a phase I/II dose finding study in patients with relapsed/refractory advanced solid tumors (ANV419-001). The primary objective of ANV419-001 is to describe the safety and tolerability of ANV419. Methods: ANV419 is administered intravenously over 15 minutes every 2 weeks, without premedication. Thirteen patients with melanoma (cutaneous (n = 3), uveal (n = 2), mucosal (n = 2), choroidal (n = 1)), renal cell carcinoma (n = 1), hepatocellular carcinoma (n = 1), colorectal cancer (n = 1), esophageal adenocarcinoma (n = 1) and adenoid cystic carcinoma (n = 1) have been dosed in six cohorts. Patients received 3mcg/kg (n = 1), 6mcg/kg (n = 1), 12mcg/kg (n = 1), 24mcg/kg (n = 4), 48mcg/kg (n = 3) 72mcg/kg (n = 3) and 108 mcg/kg (ongoing) of ANV419. Results: ANV419 is well tolerated, all related AEs are Grade 1 or Grade 2 and no DLTs have been observed. Most patients experienced chills (G1), with or without low-grade fever (G1), 2-4 hours after post- infusion, which resolved with antipyretic treatment. Two G2 AEs related to ANV419 have been reported in two patients. One patient was reported to have G2 Cytokine Release Syndrome (hypotension (G2), fever (G1), chills (G1)) and one patient experienced transient, self-limiting G2 elevation of liver function tests. Four Serious Adverse Events were reported in three patients (urinary tract infection, lethargy, thoracic pain and abdominal pain), none of which were considered related to ANV419. Pharmacodynamic evaluation on day 4 post-dosing, showed an effector cell selective, dose dependent increase of Ki-67 positive CD8 T cells (2%, 14%, 37%, 62%, 62%, vs. baseline mean (BLM) 2%) and NK cells (30%, 62%, 75%, 85%, 80%, vs BLM 6%) with a dose independent frequency of Ki67+ Tregs ranging from 4% to 25% (BLM 7%) at 3, 6, 12, 24 and 48 mcg/kg doses respectively. At 72mcg/kg mean CD8 T cell and NK proliferation was 70% and 51% respectively, while the mean Treg proliferation increased to 48%. Pharmacokinetic data (including patients treated with up to 24 mcg/kg ANV419) show a dose proportional increase of the ANV419 plasma concentration. The estimated half-life at the 24mcg/kg dose is 17.6 hrs. Four patients continue to receive ANV419. Of the 11 patients who received at least two cycles of ANV419, 4 were assessed to have stable disease. One patient progressed after 24 weeks of confirmed stable disease. Conclusions: Overall, ANV419 is well tolerated and selectively induces expansion and proliferation of CD8 T cells and NK cells, but not Tregs up to a dose of at least 48mcg/kg. Updated data will be shared during the meeting. Clinical trial information: NCT04855929.

ANV419 is an antibody-cytokine fusion protein with natural affinity to the heterodimeric IL-2Rβ/γ, but no affinity for IL-2Rα. Therefore, ANV419 preferentially stimulates CD8 T cells and NK cells over regulatory T cells. ANV419 is currently being investigated in a phase I/II dose finding study in patients with solid tumors. Goal of the presented study was the evaluation of the activity of ANV419 on NK cells and its potential combination with complementary immune-oncology mechanisms that can strengthen its NK or CD8 T cell anti-tumor response for the planned phase II trials.

BackgroundANV419 is a novel interleukin-2 (IL-2)/anti-IL-2 fusion protein with preferential signaling through the IL-2 beta/gamma receptor that induces selective proliferation of CD8 T cells and NK cells in vivo for the treatment of cancer. The safety and pharmacodynamic effects of ANV419 were studied in a 4-week cynomolgus monkey GLP study to support the ongoing PhI dose escalation clinical trial.MethodsANV419 was administered by i.v. injection over 1 min at doses of 0.03, 0.1, 0.3 mg/kg, or vehicle control on days 1 and 15 of the 29-day study. Assessments included body weight, blood pressure, hematology, clinical pathology, serum cytokines, immunophenotyping, histopathology, and pharmacokinetics.ResultsThe pharmacokinetics of ANV419 were characterized by target mediated disposition, with a half-life of approximately 24h at concentrations not affected by target mediated clearance. Dose-dependent increases in WBC were observed after each injection, driven by preferential expansion of CD8 T cells and NK cells over Tregs. NK cells were more sensitive to ANV419 than CD8 T cells reaching maximal proliferation in blood at 0.03 mg/kg vs. 0.3 mg/kg for CD8 T cells. Hematological changes included: transient dose-dependent increase in basophils; elevation in eosinophils, up to 2.2-fold above control animals at > 0.03 mg/kg, remaining within the normal range for cynomolgus monkeys (<1.94 G/L); minor decrease in platelets at day 4 after each injection. There were no relevant treatment-related changes in inflammatory serum cytokines (IL-1b, IL-5, IL-6, IL-8, IFNg, TNFa, GM-CSF). A mild systemic inflammatory response was observed at 0.3 mg/kg evidenced by a transient increase of CRP on days 4 and 19, preceded after the first injection by a slight dose dependent increase in IL-1RA at 4h post injection, and an increase in IL-10 at 24h post treatment at 0.3mg/kg. No significant changes in body weights or blood pressure and no signs of capillary leak were observed during the entire study.A multi-part PhI dose-escalation study of ANV419 has been initiated in cancer patients. In the part A single patient escalation cohort, two patients have been dosed Q2W multiple times with 0.003mg/kg and 0.006mg/kg respectively with the expected PD profile and no DLT observed.ConclusionsConsistent findings, relating to expected effects of ANV419 as a not-alpha IL-2 agonist, demonstrated a favorable tolerability and safety profile at pharmacodynamically relevant doses that strongly support its translational development in cancer patients to identify clinical benefits.

Background ANV419 is a uniquely engineered IL-2 fusion to an antibody selectively blocking the IL-2 receptor alpha (CD25) binding site. It signals selectively through the CD122/CD132 dimeric IL-2 receptor and stimulates the proliferation of CD8 T cells and NK cells while avoiding the proliferation of immunosuppressive regulatory T cells (Treg). Therefore, ANV419 has the potential to substantially separate targeted T-cell and NK cell proliferation and anti-tumor responses from the dose limiting toxicities of recombinant IL-2 (aldesleukin). ANV419 has antibody like stability and behavior and is currently in late preclinical development for tumor immunotherapy. Methods The crystal structure of ANV419 has been solved and its binding affinity to CD25 and CD122 has been determined. In vitro and in vivo studies, including pharmacodynamics and toxicity, have been performed in rodents and non-human primates. The ability of ANV419 to inhibit tumor growth has been studied in mouse syngeneic models. Results Structural analysis demonstrates that the CD25 binding site of IL-2 is completely blocked in ANV419 while the CD122/CD132 sites are available for binding. As a result, ANV419 lacks CD25 binding activity but retains IL-2 receptor beta (CD122) affinity comparable to native IL-2. In human peripheral blood monocyte cultures, ANV419 induces STAT5 phosphorylation with high selectivity for CD8 and NK cells but not Treg. Concordantly, it stimulates the proliferation of purified human CD8 T cells and NK cells but not CTLL-2 cells. A single injection of ANV419 in mice results in strong induction of the proliferation marker Ki67 specifically in CD8 T cells and NK cells but not Tregs and a selective increase of the respective cell numbers in the spleen and peripheral blood of animals. Single agent anti-tumor activity was observed in checkpoint sensitive (H22) and resistant (Renca, B16F10) syngeneic mouse tumor models. Combination of ANV419 with trastuzumab in the gastric cancer N87 xenograft model in BALB/c nude mice led to significant tumor reduction relative to trastuzumab monotherapy. In non-human primates, ANV419 is well tolerated and induces expression of Ki67 and sustained expansion in CD8 T cells and NK cells with no signs of vascular leak syndrome observed with high dose aldesleukin in patients. Conclusions The pre-clinical data suggest that ANV419 possesses a unique structure and is potent in expanding CD8 T-cells and NK cells with a marked safety window in non-human primates. This data warrants further translational development of ANV419 as an immune therapeutic in oncology.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.

An antibody to human IL-2 phenocopies CD25 and improves IL-2–based cancer immunotherapy.

The paracaspase MALT1 plays an important role in signaling pathways leading to NF-kB activation. To investigate the contribution of its proteolytic activity to overall pathway regulation we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared them to MALT1 KO animals (Malt1-/-). Malt1PD/PD mice displayed defects in multiple cell types including Treg, IL10-producing B cells and mature T and B cells. Immune defects were more pronounced in Malt1-/- animals. Both mouse lines showed abrogated B cell responses upon immunization and were protected in a Th17-dependent EAE model. Surprisingly, Malt1PD/PD animals developed a multi-organ inflammation characterized by Th2-type responses and enhanced IgG1 and IgE levels which was prevented by reconstitution with WT Treg. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and its relevance in human health and disease.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Interleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4(+) T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4(+) Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4(+) Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt.

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.

Pig-to-human xenotransplantation of islet cells or of vascularized organs would offer a welcome treatment alternative for the ever-increasing number of patients with end-stage organ failure who are waiting for a suitable allograph. The main hurdle are preexisting antibodies, most of which are specific for 'Linear-B', carbohydrate epitopes terminated by the unbranched Gal-alpha(1,3)Gal disaccharide. These antibodies are responsible for the 'hyper-acute rejection' of the xenograft by complement mediated hemorrhage. For depletion of such antibodies we have developed an artificial injectable antigen, a glycopolymer (GAS914) with a charge neutral poly-lysine backbone (degree of polymerization n = 1000) and 25% of its side chains coupled to Linear-B-trisaccharide. With an average molecular weight of 400 to 500 kD, presenting 250 trisaccharide epitopes per molecule, this multivalent array binds anti-alpha Gal antibodies with at least three orders of magnitude higher avidity on a per-saccharide basis than the monomeric epitope. In vivo experiments with non-human primates documented that rather low doses - 1 to 5 mg/kg of GAS914 injected i.v. - efficiently reduce the load of anti-Linear-B antibodies (quickly by at least 80%. This treatment can be repeated without any sensitization to GAS914. Interestingly, although the antibody levels start raising 12 h after injection, they do not reach pretreatment levels. The polymer is degraded and excreted within hours, with a minute fraction remaining in lymphoid tissue of anti-alpha Gal producing animals only, probably binding to and inhibiting antibody-producing B-cells. The results of pig-to-non-human primate xenotransplantations established GAS914 as a relevant therapeutic option for pig-to-human transplantations as well. The synthesis of GAS914 was successfully scaled up to kg amounts needed for first clinical Studies. Key was the use of galactosyl transferases and UDP-galactose for the synthesis of the trisaccharide.