Glutamate-oxaloacetate transaminase (GOT1) – fc (fragment crystallizable) fusion proteins

Abstract

The present invention refers to recombinant forms of Glutamate-oxaloacetate transaminase (GOT1) fused to a Fc region to induce a neuroprotective effect in a mammal in need thereof. The chimeric protein originated with this fusion presents an improved pharmacokinetic profile while keeping similar specific activities when compared with the standard GOT enzyme. This enhanced stability of the protein in plasma is essential for the neuroprotective role of GOT on ischemic stroke events and other pathologies.

Description

[0001] Glutamate-oxaloacetate transaminase (GOT1) - Fc (fragment crystallizable) fusion proteins

[0002] TECHNICAL FIELD

[0003] The present invention is directed to the medical field, in particular, to the treatment of brain stroke based on the administration of a recombinant form of Glutamate-oxaloacetate transaminase (GOT1).

[0004] BACKGROUND OF THE INVENTION

[0005] Glutamate oxaloacetate transaminase 1 (GOT1)

[0006] Glutamate-oxaloacetate transaminase, also known aspartate transaminase (AST), is a pyridoxal phosphate-dependent enzyme which exists in cytoplasmic and mitochondrial forms, GOT1 and GOT2, respectively.

[0007] Both forms of GOT catalyze the reversible transamination of oxaloacetate and glutamate to aspartate and a-ketoglutarate and play an essential role in amino acid metabolism in the urea and tricarboxylic acid cycles1-2(critical pathways of cell energy homeostasis), as well as in other metabolic programs.3

[0008] GOT1, the cytoplasmatic enzyme, is a homodimer polypeptide consisting of two identical monomers of 413 amino acid each, each bound to pyridoxal-5'-phosphate coenzyme (PLP).1Pyridoxal phosphate (PLP), serves as a coenzyme, facilitating the transfer of amino groups during the transamination process. In the reaction catalyzed by GOT, PLP acts as a covalently bound prosthetic group within the enzyme's active site. The amino group of aspartate initially binds to the aldehyde group of PLP, forming a Schiff base intermediate. This intermediate undergoes a proton transfer, facilitated by nearby amino acid residues in the enzyme's active site, resulting in the formation of oxaloacetate and a molecule of pyridoxamine phosphate (PMP). The a-ketogl uta rate, the other substrate in the reaction, then binds to the enzyme's active site, displacing the oxaloacetate product. This binding triggers the transfer of the amino group from the PLP-bound aspartate to a-ketoglutarate, yielding glutamate and regenerating the PLP cofactor.2The essential role of PLP for the GOT activity requires the supplementation of this cofactor when this enzyme is used as therapy.

[0009] GOT1 neuroprotective role

[0010] In clinical settings, blood GOT levels are used as indicators of liver and muscle damage or myocardial infarction.4However, based on its ability to modulate glutamate metabolism,exogenous administration of the recombinant form of GOT1 (rGOT) has been proposed as a therapy to reduce neuronal damage in pathologies such as traumatic brain injury,5post-stroke depression,6glioma,7Alzheimer's disease (AD),8amyotrophic lateral sclerosis (ALS),9and acute stroke.10 13

[0011] In the field of stroke pathology, the therapeutic mechanisms postulated to be responsible for the protective effect of rGOT are mainly associated with the metabolism and reduction of blood glutamate, which results in a lower increase in pathological glutamate caused by stroke in the cerebral parenchyma.10Other studies have also reported that GOT metabolizes brain glutamate as an energy substrate in anaerobic conditions such as ischemia.7-11This hypothesis is substantiated by the fact that the overexpression of GOT in the brain reduces the increase of glutamate and prevents the loss of ATP in ischemic conditions.14-15A recent study has also reported that mitochondrial GOT protects against energy failure after ischemia.16

[0012] The protective effect of rGOT against stroke has been mainly investigated through rodent ischemic models where intravenous rGOT administration has been associated with a reduction in ischemic lesions and better recovery.17Moreover, in the same animal models, inhibition of endogenous blood GOT activity prior to the induction of ischemic stroke resulted in more significant damage.18In two retrospective studies19-21of patients with stroke, high blood GOT activity levels were associated with good outcomes at 3 months, which suggests that exogenous administration of rGOT may be a potential stroke therapy.

[0013] A recent preclinical validation study using a new human form of GOT1 produced in E.Coli (recently accepted in iScience journal) demonstrated its protective efficacy under different experimental conditions that reflect the clinical conditions encountered during the acute phase of stroke after arterial reperfusion. These findings established that tissue neuroprotection and improved functional outcomes after stroke are unequivocally achievable with rGOT treatment. The results of tolerability in non-human primate models as well as the lack of interaction with thrombolytic therapies suggest that rGOT can be safely used immediately in patients with a suspected ischemic stroke. Cerebrospinal fluid analytics and proteomics of brain tissue, in combination with positron emission tomography imaging indicate that rGOT can also reach the brain and induce a local protective by alleviating neuronal apoptosis and induce cytoprotective autophagy. These last findings extend the protective mechanism beyond the blood-brain glutamate scanning effect initially described for GOT.22In conclusion, these findings established that rGOT might be a safe protective drug to be used in stroke patients.

[0014] This study also established that four consecutive doses of 1 mg / kg rGOT administered during the first 8 h (beginning before 2 h after arterial reperfusion) were required to induce a significantprotection effect. In this regard, new based-GOT formulations are desirable to extend the halflife of the therapy and circumvent the need for four consecutive doses of rGOT.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1: Schematic representation of the different constructs developed, including the natural GOT1 protein fused to a His tag; a GOT1 molecule fused to a mutated Fc region of a human IgGl immunoglubulin unable to dimerize and a fusion of GOT1 molecule fused to an Fc region of a human immunoglobulin forming stable dimers.

[0017] Figure 2: Analysis by PCR of viral passages. A: Baculovirus 6HisTEV GOT1 PCR1 and PCR2, table below gel indicates the samples in wells and bands size. B: Baculoviruses GOTl-GS4-Hinge Fc N297G, GOTl-GS2-Hinge Fc N297G, and GOT1-GS2 mFc 6Mut PCR1 and PCR2, tables below gels indicate the samples in wells and bands sizes.

[0018] Figure 3: Analysis of GOT1 derived proteins expression in sf9-RVN cells by SDS-PAGE. A: Protein 6HisTEV GOT1 expression analyzed by Coomassie blue staining and Western Blot anti-His-HRP. B: Proteins GOTl-GS4-Hinge Fc N297G, GOTl-GS2-Hinge Fc N297G, and GOT1-GS2 mFc 6Mut expression analyzed by Coomassie blue staining and Western Blot. Arrows indicate the recombinant proteins.

[0019] Figure 4: A: Schematic 6His TEV-GOT1 purification process. B: Schematic of purification process of GOTl-GS2-mFc 6mut, GOTl-GS2-Hinge-Fc N297G and GOTl-GS4-Hinge-Fc. Proteins were produced in insect pupae infected by the different recombinant baculoviruses (CrisBio technology). C: Analysis of proteins detected in the different steps along the purification process by using SDS-PAGE and Coomassie blue staining. The purified GOT1 proteins are observed in the eluate lane in each case.

[0020] Figure 5: The graph shows the activity levels in U / mg of the different GOT1 proteins produced in insect pupae. No significant differences among native and chimeric proteins were observed in terms of enzymatic activity in vitro.

[0021] Figure 6: A: Enzymatic activity levels in U / mg of GOTl-GS4-Hinge-Fc N297G derived from pupae inoculated with the recombinant baculovirus vector in the presence of different concentrations of PLP (0 mM, 50 mM, 75 mM and 100 mM). B: Protein GOT1 expression quantified by densitometry of Coomassie blue staining of SDS-PAGE gels resolving protein extracts from pupae co-inoculated as described in A. Proteins were also detected by anti-Fc Western BlotConcentrations of PLP of 50, 75, or 100 mM increased similarly the enzyme activity with respect to the control protein expressed in absence of PLP.

[0022] Figure 7: Comparative activity of the GOTl-GS2-mFc 6Mut, GOTl-GS2-Hinge-Fc N297G, and GOTl-GS4-Hinge-Fc N297G proteins produced or not in presence of PLP at 75 mM. In all cases, a significant increase in activity (U / mg) was observed as a result of the co-inoculation of the recombinant baculovirus in the presence of PLP.

[0023] Figure 8: The graph shows the levels of GOT1 in rats blood after injecting 1 mg / kg of purified GOT1 proteins. Fusion of GOT1 protein to a Fc immunoglobulin fragment enhanced the half-life of the enzyme with respect to the native GOT1 protein from less than 4h up to 48h in the case of the construct GOTl-GS4-Hinge-Fc N297G. Bars indicate the media of GOT1 activity in each group of rats. Proteins used in the experiment were previously conjugated with PLP.

[0024] Figure 9: Time course of blood GOT activity in ischemic rats treated with Saline (control group) and GOTl-Fc protein (SEQ ID NO 4) at a dose of 1 mg / kg, administered 60 minutes after ischemic induction.

[0025] Figure 10: Infarct size assessment in ischemic rats. Ischemic lesions are represented as % adjusted to the ipsilateral hemisphere. Saline (control group) or treatment with GOTl-Fc lmg / kg (SEQ ID NO 4) was administered as a single i.v. bolus, immediately after artery reperfusion (60 min after cerebral occlusion) in cerebral ischemic animal model. Ischemic lesions were measured by magnetic resonance imaging (MRI) on day 0 (during cerebral artery occlusion) and on days 1, 7, and 14 after ischemia induction by T2-maps. Basal lesion assessment on day 0 to confirm ischemic lesion before treatment administration.

[0026] Figure 11. Effect of rGOT treatment in a hyperglycemic model of ischemic stroke. The experimental design combines streptozotocin (STZ)-induced hyperglycemia with transient middle cerebral artery occlusion (tMCAO). Blood glucose monitoring confirms sustained hyperglycemia (>350 mg / dL) prior to ischemia. Plasma GOT activity following intravenous administration of rGOT (1 mg / kg) immediately after reperfusion shows a transient peak of approximately 4,000 U / L between 2 and 5 h. Representative magnetic resonance imaging scans and quantitative analysis illustrate infarct volumes corrected for edema. Functional evaluation using the grip strength and cylinder (laterality) tests reveals comparable motor performance and recovery trajectories between groups. Data are presented as mean ± SEM. Control HG refers to hyperglycemic control animals, and rGOTHG to rGOT-treated hyperglycemic animals.

[0027] Figure 12. A schematic overview of the clot lysis assay (CLA) and the parameters analyzed is shown. Representative temporal optical density (OD) curves illustrate clot formation and lysisinduced by rtPA (blue) or TNK (orange), assessed under co-treatment or post-treatment conditions with rGOT. Dose-response analyses of rGOT during rtPA- or TNK-mediated cotreatment display lysis rate, lysis time, and clot area, and corresponding dose-response curves are shown for post-treatment conditions. Statistical analysis was performed using ANOVA followed by Dunnett's multiple comparison test, comparing each rGOT dose with the vehicle group (n = 3-4 independent assays per condition). Bar graphs represent mean ± SD. Units of measurement are lysis rate (AU / s), lysis time (min), and clot area (AUC). The antifibrinolytic agent tranexamic acid (TXA) was included as a positive control and is indicated by a red dotted line representing its mean or median value.

[0028] Figure 13. Inhibitory effect of tranexamic acid (TXA) on rtPA- and TNK-mediated fibrinolysis. The figure illustrates the antifibrinolytic effect of tranexamic acid (TXA), used as a positive control, evaluated under co-administration and post-administration conditions with rtPA or TNK. TXA effectively inhibited fibrinolysis in all experimental settings, including rtPA or TNK cotreatment as well as post-treatment paradigms. Statistical analysis was performed using an unpaired t-test comparing TXA-treated samples with control conditions (n = 4 independent assays per group). Bar graphs represent mean ± SD.

[0029] Figure 14. Biocompatibility analysis of GOT enzymatic activity in the presence of thrombolytic agents (rtPA and TNK). GOT activity (U / L) after incubation with rtPA and TNK at different concentrations. N=3-4 independent assays per group.

[0030] Figure 15. (A) Enzymatic activity levels in U / mg of GOTl-GS4-Hinge-Fc N297G derived from pupae inoculated with the recombinant baculovirus vector in the presence of different concentrations of PLP (50, 75, 100 and 200mN). (B) Protein GOT1 expression quantified by densitometry of Coomassie blue staining of SDS-PAGE gels resolving protein extracts from pupae co-inoculated as described in A. Proteins were also detected by anti-Fc Western Blot Concentrations of PLP of 50, 75, 100 or 200 mM increased similarly the enzyme activity with respect to the control protein expressed in absence of PLP.

[0031] Figure 16. In vivo pharmacokinetic profile of GOTl-GS4-Hinge-Fc N297G with PLP co-inoculation. Healthy male rats received a single intravenous dose of 1 mg / kg GOTl-GS4-Hinge-Fc N297G produced with PLP co-inoculation. Plasma GOT activity was measured at 1, 2, 4, 6, 24, 48, and 72 hours post-injection, and at later time points up to 8 weeks. The estimated half-life was ~31 h. The dotted line indicates baseline GOT activity levels ("'100 U / L). Values displayed on top of the bars correspond to GOT activity at each analyzed time point. Data are shown as mean ± SD, with individual values represented by open circles (n = 3 per group of treatment).).SUMMARY

[0032] The present invention is directed to a fusion protein comprising at least one glutamate oxaloacetate transaminase covalently bound or linked, optionally through a peptide linker, to a Fc region (fragment crystallizable region), preferably the human IgGI Fc region or a variant thereof.

[0033] In an embodiment, the glutamate oxaloacetate transaminase is selected from the list consisting of GPT (Glutamate Pyruvate Transaminase), glutamate oxaloacetate transaminase 1 (GOT1) and glutamate oxaloacetate transaminase 2 (GOT2). Preferably, the glutamate oxaloacetate transaminase is glutamate oxaloacetate transaminase 1 (GOT1).

[0034] In an embodiment, preferably in combination with any previous or subsequent embodiment, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, or in the form of a monomeric protein construct consisting of a single polypeptide chain.

[0035] In an embodiment, preferably in combination with any previous or subsequent embodiment, the fusion protein is in the form of a homodimer, wherein each of the monomers, or polypeptide chains, of the dimer protein is a fusion protein comprising at least one glutamate oxaloacetate transaminase 1 (GOT1) covalently linked directly or optionally through a peptide linker, to a variant Fc region, preferably a variant of the human IgGI Fc region, having at least an amino acid substitution in position 297 that prevents the Fc region from being glycosylated at this site; wherein the amino acid numbering is according to the EU numbering, and wherein preferably the variant Fc region, preferably a variant of the human IgGI Fc region, is further characterized by comprising a Hinge region, preferably a Hinge mutated to improve the fragmentation resistance, preferably the mutated Hinge of SEQ ID NO 9 (DKTYTCPPCP). Preferably, each of the monomers or polypeptide chains of the dimer protein is a fusion protein having a single amino acid chain comprising SEQ ID NO: 5, or a sequence having a sequence identity of at least 90% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, preferably through a peptide linker, to the N-terminal of SEQ ID NO 7, or a sequence having a sequence identity of at least 90% with SEQ ID NO 7 that results in lack of glycosylation at position 297 of the Fc region, wherein the amino acid numbering is according to the EU numbering. More preferably, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein having the sequence set forth in SEQ ID NO 3 or SEQ ID NO 4 or a variant thereof having a sequence identity of at least 90% with SEQ ID No 3 or SEQ ID No 4 that results in lack of glycosylation at positionmore preferably, each of the monomers of the dimer protein is the sequence set forth in SEQ ID NO 4.

[0036] In an embodiment, preferably in combination with any previous or subsequent embodiment, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein comprises a single polypeptide chain having at least one glutamate oxaloacetate transaminase 1 (GOT1) covalently linked directly or optionally through a peptide linker, to a variant Fc region, preferably a variant of the human IgGl Fc region, having one or a combination of amino acid substitutions that prevents the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form. Preferably, the fusion protein is a monomeric protein having a single amino acid chain comprising: SEQ ID NO: 5, or a sequence having a sequence identity of at least 90% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, optionally through a peptide linker, to the N-terminal of SEQ ID NO 6, or a sequence having a sequence identity of at least 90% with SEQ ID NO 6 that prevents the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form, optionally through a peptide linker. More preferably, the fusion protein is in the form of a monomer protein construct having the sequence set forth in SEQ ID No 2.

[0037] In an embodiment, preferably in combination with any previous or subsequent embodiment, the fusion protein is conjugated to PLP (Pyridoxal phosphate).

[0038] In an embodiment, preferably in combination with any previous or subsequent embodiment, the fusion protein is in the form of a dimer wherein both monomers have the sequence set forth in SEQ ID NO 4, and wherein the fusion protein is conjugated to PLP (Pyridoxal phosphate).

[0039] A further aspect of the invention refers to the fusion protein according to any of the precedent embodiments, for use in therapy.

[0040] A further aspect of the invention refers to the fusion protein according to any of the precedent embodiments, for use in a method of treatment of brain stroke.

[0041] DESCRIPTION OF EMBODIMENTS

[0042] The fusion protein of the invention

[0043] Systemic treatment based on the administration of the recombinant form of transaminase Glutamate-oxaloacetate transaminase (GOT1) has been shown to induce a beneficial neuroprotective effect in animal model of stroke, measured in terms of infarct size reduction andmotor functional improvement. However, one of the bottlenecks for the future use of GOT1 as treatment for human patients is the reduced half-life of the enzyme (2 hours in rats and 5 hours in monkeys, after i.v. administration, see,22requiring multiple injections of GOT1 to maintain sustained activity in the blood and achieve the desired neuroprotective effect. Additionally, the manufacturing and purification of this protein is complex and costly using conventional methods. In the present invention, we have developed chimeric GOTl-Fc proteins [fused to different fragment crystallizable (Fc) region immunoglobulin fragments] The resulting chimeric proteins extended significantly the half-life of the enzyme in the blood (>24h) in the treated animals and improved the therapeutic functionality of the GOT1.

[0044] As shown in the in vitro functionality tests of GOT1 proteins disclosed in the examples, the functionality of the different GOT1 constructs fused to Fc were compared to the native tagged GOT1 protein. The activity units were expressed relative to the concentration of each construct (quantified by anti-GOT western blot), resulting in U / mg. All proteins, fused or not to Fc showed similar activities per mg in the in vitro assay, concluding that the fusion with the constant immunoglobulin region did not affect the GOT1 activity (Figure 5).

[0045] As shown in the / n vivo pharmacokinetic analysis of Native and Chimeric GOT1 proteins, an initial pharmacokinetic analysis of the chimeric GOTl-Fc protein was first tested in healthy experimental animals to confirm the in vivo functionality of the enzyme. The analysis was performed in male rats (Sprague-Dawley rats) (n=3) injected (i.v.) with a GOT1 protein at a dose of lmg / kg of weight to determine the activity half-life. The GOT1 presence in blood was measured at time points of 1, 2, 4, 6, 24, 48, and 72 hours post injection. The level of GOT1 in blood at lh post-injection was considered as 100% of GOT1 activity. Results clearly showed that the fusion of the Fc immunoglobulin fragment to the GOT1 protein improved the pharmacokinetic of the enzyme, extending its half-life with respect to other enzyme formulations without the Fc fragment. While the native GOT1 (produced in E.coli) activity in blood was reduced to the 50% in less than 4h (half-life 2 hours) , the chimeric GOTl-Fc proteins showed half-lives from 6 to 48h (half-life >24 hours). As shown in the figures, remarkable results are obtained when using the GOTl-GS4-Hinge-Fc N297G (SEQ ID NO 4) dimeric form.

[0046] As shown in the analysis of the neuroprotective effect of GOTl-Fc chimeric proteins in male rats (Sprague-Dawley rats) submitted to an experimental ischemic stroke, treatment with GOTl-Fc protein (GOTl-GS4-Hinge-Fc N297G (SEQ ID NO 4) at a dose of 1 mg / kg, administered 60 minutes after ischemic induction, caused a significant increase in blood GOT activity relative to basal levels (70 ± 10 U / L), with peak activities of 3,500 U / L at 2 hours. Blood GOT activity wasmaintained above 1,000 U / L for at least 24 hours after administration and returned to basal levels by day 14 (post-dose). Assessment of the ischemic lesion showed a significant reduction of infarct size and protective effect (p<0.05) in the GOT1 treated group (GOTl-GS4-Hinge-Fc N297G (SEQ. ID NO 4) compared with the control (treated with saline or vehicle) at 24 h (31,6 vs 19.7%), 7 days (22,4 vs 13.2%) and 14 days (20,1 vs 11.8%). Infarct size was defined such as percentage (%) of ischemic damage with respect to the ipsilateral hemisphere volume, corrected for brain edema. Basal lesion assessment at day 0 and before treatment administration confirmed the ischemic lesion in all the included animals and demonstrates that the infarct volume reduction observed in the GOT1 treated group was due to the therapy and no to the intrinsic variability of the surgery ischemic lesion induction.

[0047] Therefore, we can conclude that, in the context of a systemic treatment based on the administration of the recombinant form of transaminase Glutamate-oxaloacetate transaminase (GOT1) to induce a neuroprotective effect in an animal in need thereof, the GOTl-Fc chimeric proteins of the present invention reduce the need of requiring multiple injections of GOT1 to maintain sustained activity in the blood and achieve the desired neuroprotective effect as observed with the previous GOT1 version produced in E.Coli and reported elsewhere.22Consequently, a first aspect of the invention refers to a recombinant fusion protein (from hereinafter referred to as "the recombinant fusion protein of the invention" or "the fusion protein of the invention") comprising at least one glutamate modifying enzyme covalently bound or linked, optionally through a peptide linker, to a Fc region (fragment crystallizable region) or a variant thereof, preferably the human IgGl Fc region or a variant thereof.

[0048] The Fc region (fragment crystallizable region) is a portion of an antibody comprising the constant region of the heavy chains, excluding the antigen-binding domains (Fab regions). More particularly, the human IgGl Fc region is the portion of the antibody consisting of the constant domains CHj and CH3 of the heavy chain and preferably the hinge region. It does not include the variable heavy (VH) domain or the CHi domain. This region is responsible among other effects of binding to Neonatal receptor FcRn which extends the half-life of IgG by reducing lysosomal degradation.

[0049] Therefore, as used herein, the term Fc region, particularly the human IgGl Fc region, refers to a polypeptide that comprises an amino acid sequence corresponding to, or derived from, at least the CH2 and CH3 domains of a human IgGl heavy chain constant region (EU numbering positions 231-447), optionally including the hinge region (EU positions 221-230) optionally present in whole or in part. Such Fc region may include variants having one or more amino acidsubstitutions, deletions, or insertions relative to a wild-type human IgGl Fc sequence (please refer to wild type sequence indicated in SEQ ID NO 10 - 12), and may be engineered to:

[0050] • eliminate or alter N-linked glycosylation sites (e.g., by substituting N297),

[0051] • reduce or prevent dimerization (e.g., by altering residues involved in Fc homodimer interface formation),

[0052] • enhance stability or resistance to fragmentation, and / or

[0053] • confer other modifications that do not abrogate the polypeptide's derivation from the human IgGl Fc structural framework.

[0054] These variants need not retain Fc receptor binding, effector functions, or glycosylation, and may exhibit altered biophysical properties (e.g., monomeric state, modified thermal stability, reduced aggregation propensity) relative to the wild-type human IgGl Fc region, so long as the polypeptide is recognizable as being derived from and / or structurally related to the human IgGl Fc region (CH2-CH3 domains), in particular by sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% sequence identity thereto.

[0055] In particular, a wild type or canonical human IgGl Fc region (Hinge-CHz-CHa) is shown below (this sequence is numbered according to the EU numbering system which is the system followed throughout the present invention):

[0056] SEQ ID NO 10 Hinge Region (EU 221-230):

[0057] D(221) K(222) T(223) H(224) T(225) C(226) P(227) P(228) C(229) P(230)

[0058] SEQ ID NO 11 . CH2 Domain (EU 231-340):

[0059]

[0060] SEQ ID NO 12. CH3 Domain (EU 341-447):

[0061] G(341 ) Q(342) P(343) R(344) E(345) P(346) Q(347) V(348) Y(349) T(350) L(351 ) P(352) P(353) S(354) R(355) D(356) E(357) L(358) T(359) K(360) N(361) Q(362) V(363) S(364) L(365) T(366) 0(367) L(368) V(369) K(370) G(371 ) F(372) Y(373) P(374) S(375) D(376) I (377) A(378) V(379) E(380) W(381 ) E(382) S(383) N(384) G(385) Q(386) P(387) E(388) N(389) N(390) Y(391 ) K(392) T(393) T(394) P(395) P(396) V(397) L(398) D(399) S(400) D(401) G(402) S(403) F(404) F(405) L(406) Y(407) S(408) K(409) L(410) T(411 ) V(412) D(413) K(414) S(415) R(416) W(417) Q(418)Q(419)G(420) N(421)V(422) F(423) S(424) C(425) S(426) V(427) M(428) H(429) E(430)A(431) L(432) H(433) N(434) H(435) Y(436) T(437) Q(438) K(439) S(440) L(441) S(442) L(443) S(444) P(445) G(446) K(447)

[0062] Whole wild type sequence shown in SEQ ID NO 10 - 12 (SEQ ID NO 13):

[0063] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0064] It is noted that the numbering system used throughout the present invention is thus the EU numbering system. "EU numbering" is a standardized residue numbering system originally derived from the amino acid sequence of the human IgGl myeloma protein known as "Eu." In the context of human immunoglobulin heavy chains, this numbering scheme establishes a universal reference framework to identify specific positions within the antibody heavy chain, starting from the first residue of the mature N-terminus of the variable domain.

[0065] When referring to the canonical sequence of a human IgGl heavy chain, "EU numbering" means:

[0066] • The first amino acid of the variable region (after signal peptide cleavage) is designated as residue 1.

[0067] • Subsequent residues are numbered consecutively along the heavy chain, encompassing the variable (V) region, the diversity (D) segment (if applicable), the joining (J) segment, and the constant (C) regions.

[0068] Well-known structural features and functionally important residues (e.g., N297, which is a common glycosylation site) are consistently identified by their EU number, allowing researchers to unambiguously discuss and compare antibody sequences, mutations, and structural motifs. In essence, EU numbering provides a canonical, literature-standard sequence index, ensuring clarity and consistency in antibody research and engineering.

[0069] As already indicated, variants of the human IgGl Fc region are understood as an engineered, genetically modified form of the antibody's constant region in which specific amino acid substitutions and / or deletions alter its structural and functional properties. Such modifications may include:

[0070] • Prevention of Dimerization:

[0071] Changes in the Fc's CH3 domain interface that disrupt native interchain contacts, thereby preventing the Fc from forming its characteristic dimer and keeping it in a stable monomeric state.• Elimination of N-Linked Glycosylation:

[0072] Mutations at or near the canonical glycosylation site (e.g., at Asn297) that remove the consensus sequence required for carbohydrate attachment. This yields an aglycosylated Fc variant lacking the associated oligosaccharide, which typically reduces or abolishes Fc-mediated effector functions (such as binding to Fc receptors or activation of complement).

[0073] • Improved Stability and Fragmentation Resistance:

[0074] Structural adjustments that enhance the overall conformational stability of the Fc, increasing its resistance to proteolytic cleavage and fragmentation while still maintaining a monomeric form.

[0075] It is noted that to prevent Fc dimerization or to eliminate N-linked glycosylation, targeted amino acid substitutions are introduced at key interface residues or at the glycosylation site. Below are some examples and rationales for such mutations:

[0076] • Preventing Dimerization:

[0077] The Fc dimer interface involves specific residues in the CH3 domain that form intermolecular contacts. Mutating these residues to amino acids that disrupt hydrophobic interactions or sterically hinder the interface can reduce or eliminate dimer formation. For instance:

[0078] 1. T366 and L368 Mutations:

[0079] Rationale: Threonine 366 and Leucine 368 are often involved in stabilizing the CH3-CH3 interface through hydrophobic and / or polar interactions.

[0080] 2. P395 Mutation:

[0081] Rationale: Proline 395, due to its rigid backbone, can help maintain an interface geometry conducive to dimer formation.

[0082] 3. K409 Mutation:

[0083] Rationale: Lysine 409 can form stabilizing interactions (e.g., salt bridges or hydrogen bonds) across the dimer interface.

[0084] 4. M428 Mutation:

[0085] Rationale: Methionine 428 is often embedded in the hydrophobic interface region of the Fc dimer.

[0086] By selecting one or more of these substitutions, the skilled person will diminish the native Fc-Fc interactions essential for dimerization, thereby favoring a monomeric Fc fragment.

[0087] Eliminating N-Linked Glycosylation (N297):The canonical glycosylation site at Asn297 (N297) in human IgGl Fc follows the consensus motif N-X-S / T. Altering this residue removes the glycan attachment site.

[0088] N297 Mutation:

[0089] Rationale: Changing Asn297 to a residue that cannot support N-glycosylation (e.g., Ala, Gin, Gly, or Glu) breaks the N-X-S / T consensus motif.

[0090] Example Mutations: N297G, N297A, N297Qand N297E.

[0091] Any of these substitutions prevents the Fc from being glycosylated at this site. Without the carbohydrate moiety, the Fc loses much of its effector function (e.g., decreased binding to Fey receptors and impaired complement activation).

[0092] Examples of variants of the human IgGl Fc region useful in the present invention, can be selected from any of the following sequences:

[0093] SEQID NO 6 (that promotes a monomeric Fc fragment) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations capable of preventing the sequence from forming dimers; and

[0094] SEQ ID NO 8 (that prevents the Fc from being glycosylated) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations preventing the Fc from being glycosylated, preferably at the N297 site.

[0095] SEQ ID NO 6:

[0096] APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLRCHVK GFYPSDIAVE WESNGQPENN YKTTKPVLDS DGSFFLYSTL TVDKSRWQQG NVFSCSVLHE ALHNHYTQKS LSLSPGK SEQ ID NO 6 numbered in accordance with the EU numbering system:

[0097]

[0098] (361 )N (362)Q (363)V (364)S (365)L (366)R (367)C (368)H (369)V (370)K (371 )G (372)F (373)Y (374)P (375)S (376)D (377)l (378)A (379)V (380)E (381 )W (382)E (383)S (384)N (385)G (386)Q (387)P (388)E (389)N (390)N (391 )Y (392)K (393)T (394)T (395)K (396)P (397)V (398)L (399)D (400)S (401 )D (402)G (403)S (404)F (405)F (406)L (407)Y (408)S (409)T (410)L (411 )T (412)V (413)D (414)K (415)S (416)R (417)W (418)Q (419)Q (420)G (421 )N (422)V (423)F (424)S (425)0 (426)S (427)V (428)L (429)H (430)E (431 )A (432)L (433)H (434)N (435)H (436)Y (437)T (438)Q (439)K (440)S (441 )L (442)S (443)L (444)S (445)P (446)G (447)K

[0099] SEQ ID NO 8:

[0100] APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID NO 8 numbered in accordance with the EU numbering system:

[0101] CH2domain (EU 231-340):

[0102] 231 (A) 232(P) 233(E) 234(L) 235(L) 236(G) 237(G) 238(P) 239(S) 240(V) 241 (F) 242(L) 243(F) 244(P) 245(P) 246(K) 247(P) 248(K) 249(D) 250(T) 251 (L) 252(M) 253(l) 254(S) 255(R) 256(T) 257(P) 258(E) 259(V) 260(T) 261 (C) 262(V) 263(V) 264(V) 265(D) 266(V) 267(S) 268(H) 269(E) 270(D) 271 (P) 272(E) 273(V) 274(K) 275(F) 276(N) 277(W) 278(Y) 279(V) 280(D) 281 (G) 282(V) 283(E) 284(V) 285(H) 286(N) 287(A) 288(K) 289(T) 290(K) 291 (P) 292(R) 293(E) 294(E) 295(Q) 296(Y) 297(G) 298(S) 299(T) 300(Y) 301 (R) 302(V) 303(V) 304(S) 305(V) 306(L) 307(T) 308(V) 309(L) 310(H) 311 (Q) 312(D) 313(W) 314(L) 315(N) 316(G) 317(K) 318(E) 319(Y) 320(K) 321 (C) 322(K) 323(V) 324(S) 325(N) 326(K) 327(A) 328(L) 329(P) 330(A) 331 (P) 332(l) 333(E) 334(K) 335(T) 336(l) 337(S) 338(K) 339(A) 340(K)

[0103] CH3domain (EU 341-447):

[0104] 341 (G) 342(Q) 343(P) 344(R) 345(E) 346(P) 347(Q) 348(V) 349(Y) 350(T) 351 (L) 352(P) 353(P) 354(S) 355(R) 356(D) 357(E) 358(L) 359(T) 360(K) 361 (N) 362(Q) 363(V) 364(S) 365(L) 366(T) 367(C) 368(L) 369(V) 370(K) 371 (G) 372(F) 373(Y) 374(P) 375(S) 376(D) 377(l) 378(A) 379(V) 380(E) 381 (W) 382(E) 383(S) 384(N) 385(G) 386(Q) 387(P) 388(E) 389(N) 390(N) 391 (Y) 392(K) 393(T) 394(T) 395(P) 396(P) 397(V) 398(L) 399(D) 400(S) 401 (D) 402(G) 403(S) 404(F) 405(F) 406(L) 407(Y) 408(S) 409(K) 410(L) 411 (T) 412(V) 413(D) 414(K) 415(S) 416(R) 417(W) 418(Q) 419(Q) 420(G) 421 (N) 422(V) 423(F) 424(S) 425(C) 426(S) 427(V) 428(M) 429(H) 430(E) 431 (A) 432(L) 433(H) 434(N) 435(H) 436(Y) 437(T) 438(Q) 439(K) 440(S) 441 (L) 442(S) 443(L) 444(S) 445(P) 446(G) 447(K)

[0105] In a standard human IgGl Fc region, residue 297 is an Asn (N), essential for N-linked glycosylation. SEQ ID NO 8 is characterized by having Gly (G) at position 297 , indicating a site-directed mutation resulting in an Fc variant according to the invention. This N297G mutation alters the glycosylation profile and potentially affects Fc receptor binding and effector functions. SEQ ID NO 6 is characterized by having in addition to a Gly (G) at position 297 , a series of mutations that disrupt hydrophobic interactions or sterically hinder the interface thus reducingor eliminating dimer formation. In particular, the following mutations: T366R, L368H, P395K, K409T and M428L.

[0106] As used herein, the term "sequence identity" refers to the degree to which two (or more) amino acid or nucleic acid sequences are identical, on a residue-by-residue basis, over a defined comparison region (e.g., the full length of a sequence or a specified domain), when aligned for maximal correspondence. Unless otherwise specified, sequence identity is determined by comparing aligned sequences using a standard pairwise alignment algorithm (e.g., GAP, ClustalW, or BLAST) under default or recommended parameters.

[0107] The term linker, as used herein, is generally understood as short peptide sequences that occur between protein domains. Flexible linkers are often composed of one or more glycine and serine residues, a called "GS linker" so that the adjacent protein domains are free to move relative to one another. Examples of such linkers are present in any of SEQ. ID NO 3 or 4 (GS2 or GS4). In particular, as used herein, the term linker or "GS linker" refers to a flexible peptide linker sequence inserted between two or more protein domains, wherein the linker comprises one or more glycine (G) and serine (S) residues arranged in any suitable order that imparts flexibility, reduces steric hindrance, and permits the fused protein domains to adopt spatial orientations that are relatively free to move with respect to one another. Such linkers may have a length of about 2 to about 40 amino acids, and may contain primarily glycine and serine residues, optionally with other amino acids that do not substantially reduce the flexibility of the linker. By "flexible," it is meant that the linker does not impose a rigid conformation on the adjacent domains, thus facilitating proper folding, stability, and functional activity of each domain. Further examples of GS Linker Sequences:

[0108] (Gly-Ser)4: GSGSGSGS

[0109] This eight-residue linker is commonly used to connect domains in antibody fragments or fusion proteins, allowing for independent folding and reduced steric interference.

[0110] (Gly-Gly-Gly-Ser)2: GGGSGGGS

[0111] This repetitive motif of glycine and serine creates a highly flexible spacer, often employed in bispecific antibodies and single-chain variable fragments (scFvs) to enable optimal binding and reduced domain clash.

[0112] (Gly-Ser)10: A (Gly-Ser) repeat of ten units (e.g., GSGSGSGSGS...)Longer linkers composed of multiple GS repeats can be used when greater domain mobility is required, for instance, to improve enzymatic accessibility or to reduce aggregation in multidomain enzymes.

[0113] Mixed Composition GS Linkers: GSGGSGSGS

[0114] Sequences incorporating more glycine for enhanced flexibility or more serine for solubility adjustments can be tailored to achieve specific structural or functional outcomes.

[0115] In all cases, the GS linkers serve to create a polypeptide bridge of sufficient pliancy such that the protein domains connected by said linker can assume conformations conducive to their optimal function. This definition covers wild-type, engineered, and synthetic GS linkers, regardless of the production method, as long as they primarily function as flexible connectors comprising glycine and serine residues.

[0116] In a preferred embodiment of the first aspect of the invention, said at least one glutamate modifying enzyme is selected from the group consisting of a transaminase, a dehydrogenase, decarboxylase, a ligase, an aminomutase, a racemase and a transferase.

[0117] In another preferred embodiment of the first aspect of the invention, said at least one glutamate modifying enzyme is a transaminase selected from the group consisting of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, acetylornithine transaminase, ornithine-oxo-acid transaminase, succinyldiaminopimelate transaminase, 4- aminobutyrate transaminase, alanine transaminase, (s)-3-amino-2-methylpropionate transaminase, 4-hydroxyglutamate transaminase, diiodotyrosine transaminase, thyroid- hormone transaminase, tryptophan transaminase, diamine transaminase, cysteine transaminase, L-Lysine 6-transaminase, histidine transaminase, 2-aminoadipate transaminase, glycine transaminase, branched-chain-amino-acid transaminase, 5- aminovalerate transaminase, dihydroxyphenylalanine transaminase, tyrosine transaminase, phosphoserine transaminase, taurine transaminase, aromatic-amino-acid transaminase, aromatic-amino-acid-glyoxylate transaminase, leucine transaminase, 2- aminohexanoate transaminase, ornithine(lysine) transaminase, kynurenine- oxoglutarate transaminase, D-4-hydroxyphenylglycine transaminase, cysteine- conjugate transaminase, 2,5-diaminovalerate transaminase, histidinol-phosphate transaminase, diaminobutyrate-2-oxoglutarate transaminase, udp-2-acetamido-4- amino-2,4,6-trideoxyglucose transaminase and aspartate transaminase.In another preferred embodiment of the first aspect of the invention, said at least one glutamate modifying enzyme is the glutamate oxaloacetate transaminase, preferably the GPT, glutamate oxaloacetate transaminase 1 (GOT1) or the glutamate oxaloacetate transaminase 2 (GOT2). In another preferred embodiment of the first aspect of the invention, said at least one glutamate modifying enzyme is the glutamate oxaloacetate transaminase 1 (GOT1). Preferably, the said at least one glutamate oxaloacetate transaminase 1 (GOT1) comprises or consists of an amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % identical to the amino acid sequence as set forth in SEQ ID NO: 5. According to a specific embodiment, the GOT1 consists of a sequence as set forth in SEQ ID NO: 5.

[0118] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, or in the form of a monomeric protein construct comprising a single fusion protein having a single polypeptide chain.

[0119] The term dimer protein as used herein shall be understood as a type of protein that consists of two polypeptide chains, called subunits, which are typically held together by non-covalent interactions (like hydrogen bonds, ionic interactions, and hydrophobic forces) or sometimes by covalent bonds such as disulfide bridges.

[0120] The term monomeric protein as used herein shall be understood as a protein that consists of a single polypeptide chain, functioning independently without the need to pair or combine with other polypeptide chains to form its functional structure. Monomeric proteins are singleunit proteins that can perform their biological function on their own.

[0121] In another preferred embodiment of the first aspect of the invention, the Fc region of the fusion protein is a variant Fc region, preferably a variant of the human IgGl Fc region, having one or a combination of amino acid substitutions that result in lack of glycosylation and thus loss of effector function and / or prevent the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form. Said Fc region is thus preferably a human IgGl Fc region variant comprising one or a combination of amino acid substitutions, wherein said amino acid substitutions are substitutions at one or more of the following amino acids according to the EU numbering system: 297, 347, 349, 350, 351 , 352, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 405, 406, 407, 408, 409, 428 and 439.

[0122] As already indicated, variants of the Fc region, particularly variants of the human IgGl Fc region, refer to a polypeptide that comprises an amino acid sequence corresponding to, or derived from, at least the CH? and CH3 domains of a human IgGl heavy chain constant region (EU numberingpositions 231-447), optionally including the hinge region (EU positions 221-230) optionally present in whole or in part, characterized by having one or more amino acid substitutions, deletions, or insertions relative to a wild-type human IgGl Fc sequence (please refer to SEQ ID NO 10- 12), engineered to:

[0123] • eliminate or alter N-linked glycosylation sites (e.g., by substituting N297),

[0124] • reduce or prevent dimerization (e.g., by altering residues involved in Fc homodimer interface formation), and / or

[0125] • enhance stability or resistance to fragmentation.

[0126] Preferred variants according to the invention are SEQ ID NO 6 (that promotes a monomeric Fc fragment) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations capable of preventing the sequence from forming dimers; and SEQ ID NO 8 (that prevents the Fc from being glycosylated) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations preventing the Fc from being glycosylated, preferably at the N297 site.

[0127] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein comprising a variant Fc region, preferably a variant of the human IgGl Fc region, having at least an amino acid substitution in position 297 that prevents the Fc from being glycosylated at this site, such as N297G or N297E; wherein the amino acid numbering is according to the EU numbering, and wherein this substitution results in lack of glycosylation and thus loss of effector function.

[0128] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein comprising a variant Fc region, preferably a variant of the human IgGl Fc region, having at least the amino acid substitution in position 297 that prevents the Fc from being glycosylated at this site, such as N297G or N297E; wherein the amino acid numbering is according to the EU numbering, and wherein the Fc region is further characterized by comprising a Hinge region, preferably a mutated Hinge region to improve the fragmentation resistance, such as the mutated Hinge region shown in SEQ ID NO 9 or a sequence having a sequence identity of at least 90% thereto and improved fragmentation resistance.In preferred embodiments, "improved fragmentation resistance" can be demonstrated by reduced hinge cleavage or degradation in vitro or in vivo compared to a wild-type (unmodified) hinge. Such resistance may be achieved, for example, by substituting one or more residues in the hinge region with an amino acid conferring reduced susceptibility to proteolytic enzymes (e.g., replacement of a hinge-region proteolytic cleavage site with a more protease-resistant residue). Thus, a polypeptide is considered to meet the definition of having "sequence identity over a hinge region having at least 90% identity and improved fragmentation resistance" if it satisfies both the numerical threshold of >90% identity to the reference hinge sequence and includes at least one mutation that demonstrably decreases hinge fragmentation relative to the unmodified (wild-type) hinge sequence (SEQ ID NO 10). As used herein a Hinge mutated to improve the fragmentation resistance shall be understood as a flexible region that attach the protein with the Fc. Fragmentation of Fc fusion proteins, mainly occurring around the hinge regions during production, storage, and circulation in the blood. Hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially.

[0129] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein comprising at least one glutamate oxaloacetate transaminase 1 (GOT1), and wherein each of the monomers of the dimer protein is a fusion protein comprising a variant Fc region, preferably a variant of the human IgGl Fc region, having at least the amino acid substitution in position 297 that prevents the Fc from being glycosylated at this site, such as N297G or N297E, wherein the amino acid numbering is according to the EU numbering, and wherein the fusion protein further comprises a Hinge region, preferably a mutated Hinge region to improve the fragmentation resistance. Preferred variants of the IgGl Fc region according to the invention comprise or consisting of SEQ ID NO 8 (that prevents the Fc from being glycosylated) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations preventing the Fc from being glycosylated, preferably at the N297 site. It is noted that SEQ ID NO 8 can be preferably further characterized by having a Hinge region in its N terminal end, preferably a mutated Hinge region to improve the fragmentation resistance such as the mutated Hinge region of SEQ ID NO 9.

[0130] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers ofthe dimer protein is a fusion protein comprising at least one glutamate oxaloacetate transaminase 1 (GOT1), and wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein comprising a variant Fc region, preferably a variant of the human IgGl Fc region, having the sequence set forth in SEQ ID No 7 or a variant thereof having a sequence identity of at least 90% with SEQ ID No 7 that results in lack of glycosylation and thus loss of effector function and has an improved fragmentation resistance.

[0131] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein having a single amino acid chain comprising SEQ ID NO: 5, or a sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, optionally through a linker as previously defined, to the N-terminal of SEQ ID NO 7, or a sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO 7 that results in lack of glycosylation and thus loss of effector function and has an improved fragmentation resistance.

[0132] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein having a single amino acid chain comprising SEQ ID NO: 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, optionally through a linker as previously defined, to the N-terminal of SEQ ID NO 7.

[0133] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein having the sequence set forth in SEQ ID NO 3 or SEQ ID NO 4 or a variant thereof having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID No 3 or SEQ ID No 4.

[0134] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein having the sequence set forth in SEQ ID NO 3 or a variant thereof having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID No 3.

[0135] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers of the dimer protein is a fusion protein having the sequence set forth in SEQ ID NO 4 or a variantthereof having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID No 4.

[0136] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein comprises a variant Fc region, preferably a variant of the human IgGl Fc region, having one or a combination of amino acid substitutions that prevent the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form. Said Fc region is preferably a variant Fc region, preferably a variant of the human IgGl Fc region, comprising one or a combination of amino acid substitutions, wherein said amino acid substitutions are substitutions at one or more of the following amino acids according to the EU numbering system: 297, 347, 349, 350, 351 , 352, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 405, 406, 407, 408, 409, 428 and 439. More preferably, said Fc variant region, preferably a variant of the human IgGl Fc region, comprises amino acid substitutions in at least one or more of the following positions: 297, 366, 368, 395, 409 and 428 such as, but not limited to, substitutions N297G, T366R, L368H, P395K, K409T and M428L; wherein the amino acid numbering is according to the EU numbering. More preferably, said variant Fc region, preferably a variant of the human IgGl Fc region, has the sequence set forth in SEQ. ID NO 6 (that promotes a monomeric Fc fragment) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations capable of preventing the sequence from forming dimers.

[0137] As used herein, an amino acid substitution that prevents dimerization of the fusion protein refers to one or more alterations of the amino acid sequence of the fusion protein at a position involved in protein-protein interaction, wherein the substitution disrupts the dimer interface and thereby reduces or eliminates the ability of the fusion protein to form stable dimers, resulting in the maintenance of said fusion protein predominantly in a monomeric form. Such substitutions may include, but are not limited to, replacing a hydrophobic residue at the dimer interface with a charged or polar residue, or introducing a sterically hindered residue that disrupts inter-protein contacts. By way of non-limiting example, substituting leucine (L) or valine (V) at a designated interface position with charged residues such as glutamate (E) or lysine (K), or a bulky residue such as tryptophan (W), can prevent or significantly reduce dimer formation and thus stabilize the fusion protein in a monomeric configuration.

[0138] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein comprises at least oneglutamate oxaloacetate transaminase 1 (GOT1), and wherein the fusion protein further comprises a variant Fc region, preferably a variant of the human IgGl Fc region, having one or a combination of amino acid substitutions that prevent the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form. Said Fc region is preferably a variant of the human IgGl Fc region comprising one or a combination of amino acid substitutions, wherein said amino acid substitutions are substitutions at one or more of the following amino acids according to the EU numbering system: 297, 347, 349, 350, 351 , 352, 354, 356, 357, 360, 362, 364, 366, 368, 370, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 405, 406, 407, 408, 409, 428 and 439. More preferably, said Fc variant region of the human IgGl Fc fragment comprises amino acid substitutions in at least one or more of the following positions: 297, 366, 368, 395, 409 and 428 such as, but not limited to, amino acid substitutions: N297G, T366R, L368H, P395K, K409T and / or M428L; wherein the amino acid numbering is according to the EU numbering. More preferably, said variant Fc region, preferably a variant of the human IgGl Fc region, comprises or consists of the sequence set forth in SEQ ID NO 6 (that promotes a monomeric Fc fragment) or any further sequences sharing at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations capable of preventing the sequence from forming dimers. Optionally, SEQ ID NO 6 can be further characterized by having a Hinge region covalently linked to its N terminal, preferably a mutated Hinge region to improve the fragmentation resistance such as the Hinge region defined by SEQ ID NO 9.

[0139] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein is a monomeric protein having a single amino acid or polypeptide chain comprising SEQ ID NO: 5, or a sequence having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, optionally through a linker as defined above, to the N-terminal of SEQ ID NO 6, or a sequence having a sequence identity of at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity thereto characterized by having one or more mutations capable of preventing the sequence from forming dimers.

[0140] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein is a monomeric protein having a single amino acid chain comprising SEQ ID NO: 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked to the N-terminal of SEQ ID NO 6, optionally through a peptide linker.In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein is the sequence set forth in SEQ ID No 2 or a variant thereof having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID No 2.

[0141] In another preferred embodiment of the first aspect of the invention, the fusion protein is in the form of a monomer protein construct, wherein the fusion protein is the sequence set forth in SEQ ID No 2.

[0142] Conjugation of the fusion protein of the invention to PLP

[0143] On the other hand, it is noted that glutamate-oxaloacetate transaminase, also known as aspartate transaminase, is a pyridoxal phosphate-dependent enzyme. Pyridoxal phosphate (PLP), as well known as Vitamin B6, serves as a coenzyme, functioning as a cofactor that facilitates the transfer of amino groups during the transamination process. Therefore, the activity of the enzyme is highly dependent on its conjugation with PLP. Therefore, in a further embodiment of the invention, the fusion protein as defined in any of the previous embodiments of the first aspect of the invention is preferably conjugated to PLP or to a precursor of this active coenzyme such as pyridoxal. Preferably, the fusion protein as defined in any of the previous embodiments of the first aspect of the invention as comprising at least one glutamate oxaloacetate transaminase, preferably at least one glutamate oxaloacetate transaminase 1 (GOT1), is preferably conjugated to PLP or to a precursor of this active coenzyme such as pyridoxal. It is noted that this specific embodiment refers to the fusion protein as defined in any of the previous embodiments of the first aspect of the invention, conjugated to PLP, independently of the method of conjugation, which could be any method including, but not limited to, the in vivo method explained later in the present specification; that is, this specific embodiment refers to the result of the conjugation along with the transaminase synthesis in presence of vitamin B6 forms such as PLP, pyridoxine or pyridoxal.

[0144] It is noted that, as already indicated, structurally, hGOTl is characterized as a homodimeric (a2) enzyme where each monomer consists of a large and small domain. The small domain of the enzyme is shaped by four a-helices, three p-strands, and the N- and C- termini. The large domain consists of a 7-stranded p-sheet and several short a helices, which we refer to as the large domain core. The large domains from each monomer form the dimer interface, and two PLP binding sites are located at this interface. PLP is stabilized by surrounding residues, where the D223 residue acts as an anchor by forming a hydrogen bond with the positively charged nitrogenin the pyridine of PLP. The indole ring of W141 participates in both n-n aromatic stacking and hydrophobic interactions with the pyridine of PLP. These interactions place the coenzyme for formation of the covalent Schiff base linkage with K259 residue. The phosphate group of PLP is stabilized by G109, T110, S256, S258, and R267 of the same monomer as well as T71 from the opposite monomer. The two primary substrate binding residues are R387 and R293 and are located at the entrance of the binding site.

[0145] Thus, as used herein by "conjugated to PLP" shall be understood as the combination of the protein part of the enzyme, also known as apoenzyme, with the non-protein part of the enzyme, known as coenzyme, to form the active form of the enzyme, also known as holoenzyme. This combination of the parts of both parts of the holoenzyme is highly dependent on Pyridoxal phosphate availability and can take place inside the cell. Highly active GOT holoenzyme has been reported to be purified in a complex process including an in vitro heat treatment at 709C in presence of a-ketoglutarate and Pyridoxal phosphate (patent W02016157190A1), which may contribute to apoenzyme-PLP conjugation process.

[0146] GOT is an essential enzyme involved in amino acid metabolism, particularly in the reversible conversion of glutamate and oxaloacetate to aspartate and a-ketoglutarate, through the transference of an amino group. This enzymatic activity is pivotal in various physiological processes, including the Krebs cycle and the biosynthesis of amino acids.

[0147] Pyridoxal phosphate (PLP), serves as a coenzyme, functioning as a cofactor that facilitates the transfer of amino groups during the transamination process. In the reaction catalyzed by AST, PLP acts as a covalently bound prosthetic group within the enzyme's active site. The amino group of aspartate initially binds to the aldehyde group of PLP, forming a Schiff base intermediate. This intermediate undergoes a proton transfer, facilitated by nearby amino acid residues in the enzyme's active site, resulting in the formation of oxaloacetate and a molecule of pyridoxamine phosphate (PMP). The a-ketoglutarate, the other substrate in the reaction, then binds to the enzyme's active site, displacing the oxaloacetate product. This binding triggers the transfer of the amino group from the PLP-bound aspartate to a-ketoglutarate, yielding glutamate and regenerating the PLP cofactor. The role of PLP for the GOT activity thus significantly improves with the supplementation of this cofactor when this enzyme is used as therapy. This is clearly illustrated in the present invention as explained below.

[0148] As shown in the example, inoculation of the baculovirus vector expressing the fusion protein of the invention in presence of PLP at 50 mM, 75 mM, and 100 mM increased GOT1 activity in vitro by more than four times compared to the extract without PLP (0 mM). Apparently, rangesbetween 50-100 mM were found as the best conditions for increasing GOT1 activity, suggesting the best conjugation of the enzyme and maintaining optimal GOT1 productivities. Moreover, coinoculation experiments (PLP and baculovirus vector) were carried out in pupae infected with baculoviruses expressing the GOTl-GS4-Hinge-Fc N297G construct, with the aim of integrating PLP supplementation directly into the enzyme biosynthesis process using increasing PLP concentrations (0-200 mM). Co-inoculation with PLP at 50 mM, 75 mM, and 100 mM increased GOT1 activity in vitro by more than four times compared to the extract without PLP, while maintaining protein expression levels and solubility comparable to the control condition (Figure 15A). In contrast, higher PLP concentrations (200 mM) not only failed to provide further improvement but also resulted in a complete loss of activity. These findings suggest a potential toxic effect on one or more components of the expression system, either the baculovirus or the insect cells present in Trichoplusia ni pupae. Western blot analysis confirmed proper expression, and activity assays demonstrated that in vitro conjugation with PLP was consistently achieved across all recombinant GOTl-derived proteins tested (Figure 15B). The same co-inoculation strategy was applied to additional GOT1 constructs. In all cases, a significant increase in activity (U / mg) was observed as a result of the co-inoculation of the recombinant baculovirus in the presence of PLP. These findings indicate that the co-supply of PLP during expression not only enhances GOT1 enzymatic performance but also represents a broadly applicable strategy for the stabilization and functional optimization of GOTl-based fusion proteins.

[0149] In addition, to validate the effect of co-inoculating PLP during protein synthesis, blood GOT activity was measured in healthy male rats after intravenous administration of 1 mg / kg of the GOTl-GS4-Hinge-Fc N297G construct, selected among the four synthesized constructs for its superior plasma half-life and activity. Blood samples were collected at 1, 2, 4, 6-, 24-, 48-, and 72-hours post-injection, and at later time points up to 8 weeks, to measure plasma GOT activity. GOT activity increased sharply within the first hours, peaking at ~4,300 U / L at 1 h and remaining above 3,000 U / L up to 6 h post-dose. Enzymatic activity declined gradually thereafter, reaching ~l,700 U / L at 24 h and ~774 U / L by day 4 (Figure 16). At 1 week, activity remained at ~500 U / L and decreased progressively to basal values (~100 U / L) by the third week post-injection. Pharmacokinetic analysis revealed an estimated plasma half-life of ~31 h and confirmed that PLP co-inoculation enhanced both the stability and catalytic functionality of the recombinant enzyme in circulation compared with the non-PLP counterpart.

[0150] Bringing together these results, it is established a relationship between increasing amounts of vitamin B6 forms available for the cells (such as PLP, Pyridoxal or Pyridoxine) and the specific activity of the purified enzymes, due to a higher proportion of holoenzyme formation.Once determined the optimal PLP concentration for co-inoculations, the other GOT1 constructs were produced in the same conditions and analyzed using Western blot and activity tests. The figures show that the in vivo GOT1-PLP conjugation was achieved in all recombinant GOT1-derived proteins analyzed.

[0151] As further shown in the example, pharmacokinetic analysis of the chimeric GOTl-Fc protein was tested in healthy experimental animals to confirm the in vivo functionality of the enzyme. The analysis was performed in male rats (n=3) treated with a GOT1-GS4-Hinge-Fc N297G protein conjugated in pupa (75mM PLP). Treatment administration was performed through the i.v. route through the tail vein, and each tested dose was adjusted to a final volume of 1 mL. Blood samples for GOT and glutamate analyses were collected from the tail vein into test tubes (BD Microtainer K2E Tubes, Franklin Lakes, New Jersey, USA). The protein dose was of lmg / kg of weight to determine the half-life of the protein in blood. With this experiment we wanted to demonstrate the half-life of the insect-derived GOT1 in blood and how it compares with a control native GOT1. The GOT1 activity in blood was measured at time points of 1, 2, 4, 6, 24, 48, and 72 hours post injection. The level of GOT1 in blood at lh post-injection was considered as 100% of GOT1 activity. GOT activity in the blood was measured using the GOT / AST activity test (Biosystems Biotech Spain). Results clearly showed that the fusion of the Fc immunoglobulin fragment to the GOT1 protein improved the pharmacokinetic of the enzyme, extending its halflife. While the native GOT1 activity in blood was reduced to 50% in less than 4h, the chimeric GOTl-Fc protein showed a half-life up to 48h post inoculation. In vivo PLP conjugation to GOT1 by the addition of PLP to the pupae during the inoculation with baculovirus did not affect the half-life of the protein in blood. Differences in half-life are due to the incorporation transcriptionally of a Fc immunoglobulin fragment to the enzyme.

[0152] In summary, supplementation of the production medium with vitamin B6 forms such as PLP, pyridoxine or pyridoxal significantly enhances the in vitro potency of the fusion protein purified. Such improvement of GOT activity is accompanied by a significant increase in half-life when the Fc immunoglobulin fragment is incorporated to the enzyme. Such increase is especially remarkable when the GOTl-GS4-Hinge-Fc N297G is used.

[0153] Method of production of the recombinant fusion protein of the invention.

[0154] The present invention further provides methods for producing a recombinant fusion protein of the invention, as defined in the first aspect and related embodiments. The methods described herein employ molecular biology, genetic engineering, and related production techniques known in the art to achieve expression and scalable yield of the recombinant fusion protein.The present invention contemplates the use of any suitable host cell system to produce the recombinant fusion protein. Without limiting the scope, host cells may include bacterial, yeast, fungal, mammalian, or plant cells, as well as insect cell lines known in the art for heterologous gene expression (for example, Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells). In one preferred embodiment, the recombinant fusion protein of the invention is expressed in insect cell lines derived from the Lepidopteran genus Trichoplusia, particularly Trichoplusia ni cells, which are well-suited for the scalable production of recombinant proteins.

[0155] In addition to cell culture-based production systems, the present invention further provides methods for producing the recombinant fusion protein in living insects, thereby harnessing the insect's natural capacity as a bioreactor or "biofactory." More specifically, in one aspect of the invention, the recombinant fusion protein is produced within insect pupae or larvae. It has been found that pupae, and in particular those belonging to the order Lepidoptera, such as those of the genera Trichoplusia, Rachiplusia, or Bombyx, serve as an efficient and sustainable means to yield the desired recombinant fusion protein. In a particularly preferred embodiment, Trichoplusia ni pupae are employed as living biofactories.

[0156] As already indicated, to achieve reproducible and scalable production of the recombinant fusion protein of the invention, cell culture-based systems may be employed, utilizing either suspension or adherent cell lines. Insect cell lines— such as Sf9, Sf21, or Tni cells— are commonly used for baculovirus expression systems, providing high yields of correctly folded and post-translationally modified recombinant proteins. The baculovirus-insect cell platform is well-established, allowing for rapid scale-up from small shake flasks to larger bioreactors with volumes ranging from a few liters to several thousand liters.

[0157] Beyond insect systems, the invention also contemplates the use of alternative eukaryotic expression systems, including mammalian cell lines. Mammalian host cells, such as Chinese Hamster Ovary (CHO) cells, HEK293 cells, or NSO cells, are frequently employed when humanlike post-translational modifications (such as glycosylation patterns) are desired or required to ensure proper protein function and stability. These cell lines can be adapted for serum-free, suspension growth, allowing for large-scale cultivation in stirred-tank bioreactors. Techniques such as transient transfection or the generation of stable cell lines can be employed, depending on the production timelines, yield requirements, and regulatory considerations.

[0158] Similarly, yeast-based systems (e.g., Pichia pastoris, Saccharomyces cerevisiae) or fungal and plant expression platforms can be considered, especially when cost-effectiveness and straightforward scalability are priorities. Each platform can be selected, optimized, and scaledaccording to the specific requirements of the reco binant fusion protein, such as proper folding, yield, and post-translational modifications.

[0159] The following are exemplary embodiments illustrating how the recombinant fusion protein can be produced, isolated, and purified from the chosen expression systems:

[0160] Production in Insect Cells or Insect Pupae:

[0161] Inoculation and Expression: A recombinant baculovirus encoding a fusion protein of the invention is used to infect insect cell cultures or directly infect insect pupae or larvae. Under optimal conditions (e.g., temperature, pH, nutrient concentrations), the infected cells or insects synthesize and accumulate the fusion protein.

[0162] Harvesting: In cell culture systems, the culture medium and cells are harvested when maximum protein expression is reached— typically a few days post-infection. For insect pupae or larvae, the biomass is collected at the appropriate time post-infection when target protein levels are maximal.

[0163] Production in Mammalian Cells:

[0164] Transient Transfection: Plasmid DNA encoding the recombinant fusion protein of the invention is transfected into HEK293 or CHO cells, typically using cationic lipids, electroporation, or other established methods. Cultivation under optimized conditions leads to transient expression of the recombinant protein in the conditioned medium.

[0165] Stable Cell Line Generation: Alternatively, the gene encoding the fusion protein can be stably integrated into the host cell genome. After selecting high-producing clones, large-scale batch or fed-batch bioreactor cultivation is performed to accumulate the recombinant protein in the medium.

[0166] Isolation and Purification:

[0167] Regardless of the chosen host system, once the target protein is expressed, it is isolated and purified by methods known in the art to achieve high purity and activity. These may include:

[0168] • Cell Lysis and Clarification (if intracellular): Harvested cells or insect biomass are disrupted by mechanical, chemical, or enzymatic means. The crude lysate is then clarified by centrifugation or filtration to remove cell debris.

[0169] • Initial Capture Step: Affinity chromatography (e.g., using tags engineered into the fusion protein), ion exchange chromatography, or other selective binding methods are used to capture the target protein from the clarified lysate or conditioned medium.• Intermediate and Polishing Steps: Additional chromatographic methods (e.g., size exclusion, reverse-phase, hydrophobic interaction) may be employed to remove contaminants, aggregate species, or host cell proteins, resulting in a final purified recombinant fusion protein.

[0170] • Buffer Exchange and Formulation: The purified protein is subsequently subjected to buffer exchange, concentration, and formulation steps, ensuring stability and suitability for therapeutic applications.

[0171] Thus, the invention supports flexible production strategies using a wide range of biological systems. By selecting the appropriate host cell type, expression method, and purification scheme, the recombinant fusion protein can be reliably produced in a scalable manner to meet the requirements of industrial, therapeutic, diagnostic, or research applications.

[0172] Therefore, in a second aspect of the invention, the invention refers to a method of producing the recombinant fusion protein of the invention (from hereinafter "method of production of the recombinant fusion protein of the invention") comprising providing a host expression system, introducing a nucleic acid sequence encoding the recombinant fusion protein into said host system, cultivating the host under conditions conducive to protein expression, and subsequently preferably isolating and purifying the recombinant fusion protein.

[0173] In an embodiment of the second aspect of the invention, the method of the invention further comprises selecting a host from the group consisting of bacterial, yeast, fungal, mammalian, plant, and insect cells, or living insect pupae or larvae, based on desired protein yield, quality, and post-translational modifications.

[0174] In another embodiment of the second aspect of the invention, the host system is an insect cell line, such as Trichoplusia ni or Spodoptera frugiperda cells, introducing a nucleic acid sequence encoding the recombinant fusion protein, preferably by infection with a recombinant baculovirus vector encoding the fusion protein. The method includes cultivating the infected cells in suspension culture, harvesting the cells or supernatant at peak expression, and purifying the recombinant fusion protein.

[0175] In another embodiment of the second aspect of the invention, the host comprises insect pupae of the genus Trichoplusia, Rachiplusia, or Bombyx. The method includes inoculating pupae with a recombinant baculovirus encoding the fusion protein of the invention, cultivating the pupae under suitable conditions, and harvesting the pupae once the fusion protein is produced. The protein is then extracted and preferably purified by chromatographic methods.In another embodiment of the second aspect of the invention, the host system is a mammalian cell line such as CHO or HEK293 cells. The method includes transiently or stably expressing the fusion protein of the invention, scaling up production in a bioreactor, and purifying the protein from cell culture supernatant using affinity and size exclusion chromatography. Preferably, the mammalian cells are adapted to serum-free suspension culture, thereby facilitating large-scale production and simplifying downstream purification.

[0176] In another embodiment of the second aspect of the invention, the host is a yeast strain, such as Pichia pastoris, engineered to express the recombinant fusion protein of the invention under an inducible promoter. Following induction, the fusion protein of the invention is secreted into the medium, harvested, and purified using affinity and ion exchange chromatography.

[0177] In another embodiment of the second aspect of the invention, the host is a plant expression system (e.g., transient expression in Nicotiana benthamiana leaves). The method includes infiltrating plant tissues with an expression construct, cultivating under suitable conditions, harvesting the biomass, and isolating the recombinant fusion protein through clarification and chromatography steps.

[0178] In addition, the present invention further provides products especially useful for the expression of the recombinant fusion proteins of the invention.

[0179] That is, in a third aspect, the present invention provides for a nucleic acid sequence (from hereinafter nucleic acid of the invention) encoding the fusion protein as defined in any of the embodiments of the first aspect of the invention.

[0180] In a fourth aspect, the present invention provides for a vector (from herein after vector of the invention) comprising one or more of the nucleic acid sequences of the invention. The vector can also be referred to as a "transfer vector". The skilled person will recognize and select suitable vectors for the expression of fusion proteins. In one embodiment according to the technology, the fusion protein is expressed under the control of a suitable promoter, such as a polyhedrin promoter.

[0181] In a fifth aspect, the present invention provides for a bacmid (from hereinafter bacmid of the invention) comprising the nucleic acid sequence and / or the vector of the invention, preferably the bacmid comprises a mini Tn7-replicon. The term "mini Tn7-replicon" is well-known in the art and will be understood as sequence needed for incorporation of foreign sequences into an empty bacmid.As used herein, a "bacmid" refers to a plasmid construct which contains the DNA sequence sufficient for generating a baculovirus when transfected into a cell or insect.

[0182] The transfer vector and / or bacmid of the invention may be derived from any of the commercially available baculovirus expression systems "Bac-to-Bac®" (invitrogen™), "BacPAKTM" (ClontechTM), "FlashBACTM" (Oxford Expression TechnologiesTM), "BacuVanceTM" (GenScriptTM), "Bac-N-Blue DNATM" (invitrogenTM), "BaculoDirectTM" (invitrogenTM), "BacVector®" 1000, 2000, 3000 (Novagen®), "DiamondBacTM" (Sigma-Aldrich®) or "BaculoGoldTM" (BD biosciencesTM). In one embodiment, the present invention provides for a bac-to-bac technology that can be used to obtain the baculovirus by transposition. The "bac-to-bac technology" is well-known in the art and is generally understood as a method used for the production of recombinant proteins, particularly in insect cells. It involves the use of a baculovirus expression system, which utilizes the baculovirus AcMNPV (Autographa californica multiple nucleopolyhedrovirus) to deliver and express foreign genes in host cells, such as insect cells including but not limited to Sf9 or Sf21.

[0183] In a sixth aspect, the present invention provides for a baculovirus (from hereinafter baculovirus of the invention) comprising the nucleic acid sequence and / or the vector and / or the bacmid of the invention. In a preferred embodiment, the baculovirus is derived from AcMNPV (Autographa californica nuclear polyhedrosis virus) or BmNPV (Bombyx mori nucleopolyhedro virus).

[0184] On the other hand, the baculovirus expression vector system (BEVS) is a well-established method for the production of recombinant proteins, for example proteins to be used as vaccines, therapeutic molecules or diagnostic reagents. With its potential for over-expression and rapid speed of development, the BEVS is one of the most attractive choices for producing recombinant proteins for any purpose. The most employed baculovirus vector used in industry for recombinant protein expression is based on Autographa californica multinuclear polyhedrosis virus (AcMNPV) with Spodoptera frugiperda 9 (Sf9) or 21 (Sf21) insect cells as suitable expression hosts,23as well as Trichoplusia ni (T. ni) insect larvae as living biofactories.24Since the BEVS was developed in the 80's,25hundreds of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Recently, new baculovirus vectors have been described. For instance, WO 2012 / 168493 and WO 2012 / 168492 relate to recombinant DNA elements for the expression of recombinant proteins in insects and insect cells. Specifically, WO 2012 / 168492 relates to recombinant baculovirus, transfer vectors, and bacmids suitable for use in the present invention.In the context of the present invention, any conventional baculovirus can be used for the expression of the fusion proteins as defined in the first aspect of the invention. For example, any improved baculovirus can be used, such as to Top-Bac. The term "Top-Bac" is well-known in the art and generally refers to a refined version of the bac-to-bac technology used for the expression of recombinant proteins in e.g., insect cells via a baculovirus system.

[0185] In a seventh aspect, the present invention provides for a host cell as defined in the second aspect of the invention comprising the nucleic acid sequence and / or the vector and / or the bacmid and / or the baculovirus of the invention. Preferably, the host cell is an insect cell, preferably a Spodoptera frugiperda 9 (Sf9), 21 (Sf21), Bm5 or BmE-SWU insect cell.

[0186] In a further aspect, the eight aspect, the invention provides for an insect comprising the nucleic acid sequence and / or the vector and / or the bacmid and / or the baculovirus and / or the host cell of the invention. In a preferred embodiment according to the technology, the insect is a pupa or a larva. For instance, WO2017 / 046415 describes the use of a pupa. In one embodiment according to the technology, the pupa belongs to the order Lepidoptera, preferably to the genus Trichoplusia, Rachiplusia or Bombix mori, even more preferably to the genus Trichoplusia and to the species Trichoplusia ni. In the context of the present invention, when the pupa belongs to the genus Trichoplusia or Rachiplusia, a AcMNPV baculovirus is preferably used. In the case the pupa belongs to the genus Bombix mori, a BmNPV baculovirus is preferably used.

[0187] In an embodiment, the method comprises the following steps:

[0188] a. infecting, transfecting, transducing or transforming an isolated host with a composition comprising:

[0189] i. a recombinant baculovirus, vector, or nucleic acid sequence, comprising a nucleic acid sequence encoding a recombinant protein operably linked to a promoter, wherein the recombinant protein is a PLP dependent enzyme preferably a fusion protein as defined in any of the embodiments, or combinations thereof, of the first aspect of the invention, and

[0190] b. extracting and optionally purifying the recombinant protein by conventional means, preferably by combining affinity chromatography with protein A resin with other chromatographic and filtration steps in a process similar to monoclonal antibodies purifications.

[0191] Method for producing the conjugated fusion protein of the invention.As already indicated, the fusion protein of the invention can be preferably conjugated to PLP. In this sense, such a conjugated fusion protein, in another embodiment, can be manufactured by a method generally comprising the following steps:

[0192] 1. Providing a Host Expression System:

[0193] As already indicated for the general method already described, a suitable host system may be employed, including but not limited to bacterial, yeast, fungal, mammalian, plant, or insect cellbased expression systems, as well as living insect pupae or larvae.

[0194] 2. Introducing a Nucleic Acid Sequence Encoding the Recombinant Fusion Protein:

[0195] a) A nucleic acid construct that encodes the recombinant fusion protein to be conjugated is introduced into the host system. This may involve:

[0196] o Transformation or Transfection: In the case of bacterial, yeast, or mammalian cells, the vector harboring the gene of interest is introduced via chemical methods, electroporation, or viral vectors.

[0197] o Viral Infection: For insect cell cultures or pupae, a recombinant baculovirus vector may be used to deliver and express the fusion protein gene.

[0198] 3. Cultivating the Host Under Conditions Conducive to Protein Expression:

[0199] The host is grown or maintained under optimal conditions (e.g., temperature, pH, oxygenation, nutrients) to allow for the expression of the fusion protein. These conditions may vary depending on the host system employed.

[0200] 4. Conjugation in the Presence of PLP:

[0201] The method (the method of producing the conjugated fusion protein of the invention) is characterized by being carried out in the presence of PLP, ensuring that the conjugation occurs as the protein is expressed and / or processed. The presence of PLP can be achieved by:

[0202] • Simultaneous Addition: Including PLP in the culture medium from the outset or coinoculating with the PLP jointly with the baculovirus vector into the insect pupa, allowing the newly synthesized fusion protein to interact and form a conjugate with PLP during the expression phase; or

[0203] • Subsequent Addition: Introducing PLP into the culture medium ata later stage (e.g., midlog phase or after a certain accumulation of the fusion protein) to control the extent or timing of conjugation.5. Isolation and Purification of the Conjugated Fusion Protein:

[0204] After the fusion protein has been expressed and conjugated to PLP, the biomass (cells, pupae, or plant material) or culture supernatant is harvested. Standard protein purification techniques are applied, which may include:

[0205] • Clarification: Removing cellular debris through centrifugation or filtration.

[0206] • Initial Capture Step: Using affinity chromatography (if tags are present), ion exchange chromatography, or other selective binding steps that distinguish the conjugated fusion protein from host proteins.

[0207] Polishing Steps: Further purification steps such as size exclusion chromatography or hydrophobic interaction chromatography to remove contaminants, aggregates, or unbound PLP.

[0208] Formulation: Exchanging buffers, adjusting concentration, and sterilizing if necessary to obtain the purified conjugated fusion protein in a stable and application-ready form.

[0209] Therefore, in a nineth aspect of the invention, the invention refers to a method of producing the recombinant PLP conjugated fusion protein of the invention (from hereinafter "method for Producing the Conjugated Fusion Protein of the invention") comprising providing a host expression system, introducing a nucleic acid sequence encoding the recombinant fusion protein into said host system, cultivating the host under conditions conducive to protein expression, and subsequently preferably isolating and purifying the recombinant fusion protein, wherein the method is carried out in the presence of vitamin B6 forms such as PLP, pyridoxine or pyridoxal, ensuring that the conjugation occurs as the protein is expressed and / or processed, preferably the method is carried out in the presence of a concentration of PLP of less than 200 mM, preferably in the presence of any concentration greater than 0 mM and less than 200 mM, more preferably between 20 and lOOmM, more preferably between 50 mM and 100 mM.

[0210] In a preferred embodiment of the nineth aspect of the invention, the method of the invention further comprises selecting a host from the group consisting of bacterial, yeast, fungal, mammalian, plant, and insect cells, or living insect pupae or larvae, based on desired protein yield, quality, and post-translational modifications.

[0211] In another preferred embodiment of the nineth aspect of the invention, the host system is an insect cell line, such as Trichoplusia ni or Spodoptera frugiperda cells, introducing a nucleic acid sequence encoding the recombinant fusion protein, preferably by infection with a recombinant baculovirus vector encoding the fusion protein. The method includes cultivating the infectedcells in suspension culture, harvesting the cells or supernatant at peak expression, and purifying the recombinant fusion protein.

[0212] In another preferred embodiment of the nineth aspect of the invention, the host comprises insect pupae of the genus Trichoplusia, Rachiplusia, or Bombyx. The method includes inoculating pupae with a recombinant baculovirus encoding the fusion protein of the invention, cultivating the pupae under suitable conditions, and harvesting the pupae once the fusion protein is produced. The protein is then extracted and preferably purified by chromatographic methods. In another preferred embodiment of the nineth aspect of the invention, the host system is a mammalian cell line such as CHO or HEK293 cells. The method includes transiently or stably expressing the fusion protein of the invention, scaling up production in a bioreactor, and purifying the protein from cell culture supernatant using affinity and size exclusion chromatography. Preferably, the mammalian cells are adapted to serum-free suspension culture, thereby facilitating large-scale production and simplifying downstream purification.

[0213] In another preferred embodiment of the nineth aspect of the invention, the host is a yeast strain, such as Pichia pastoris, engineered to express the recombinant fusion protein of the invention under an inducible promoter. Following induction, the fusion protein of the invention is secreted into the medium, harvested, and purified using affinity and ion exchange chromatography. In another preferred embodiment of the nineth aspect of the invention, the host is a plant expression system (e.g., transient expression in Nicotiana bentham iana leaves). The method includes infiltrating plant tissues with an expression construct, cultivating under suitable conditions, harvesting the biomass, and isolating the recombinant fusion protein through clarification and chromatography steps.

[0214] In summary, the method of producing a conjugated fusion protein of the invention involves expressing the recombinant fusion protein in a chosen host system in the presence of vitamin B6 forms such as PLP, pyridoxine or pyridoxal to achieve conjugation. The process can range from entirely in vivo conjugation to hybrid strategies that involve post-harvest modifications. On the other hand, as already indicated, the method of the nineth aspect is carried out in the presence of vitamin B6 forms such as PLP, pyridoxine or pyridoxal under conditions that ensure conjugation occurs as the recombinant protein is expressed and / or processed. Vitamin B6 forms such as PLP, pyridoxine or pyridoxal, can be added to the production system at any point during the recombinant protein production process; however, it is preferably added simultaneously with the entity encoding the recombinant protein (e.g., a nucleic acid, expression vector, or recombinant baculovirus). In this manner, vitamin B6 forms such as PLP, pyridoxine or pyridoxal,is present at or shortly after the onset of protein expression, thereby facilitating efficient conjugation.

[0215] In this sense, in the examples of the present invention, we describe for the first time a simple methodology to conjugate PLP and GOT1 in Trichoplusia ni insect pupae co-injected with PLP jointly with a recombinant baculovirus expressing the GOT1 protein under certain parameters. The resulting pupa-derived GOT1 was naturally conjugated with PLP without further processing, increasing its activity significatively with respect to the non-conjugated enzyme and showing similar enzymatic activity to the protein conjugated with PLP by heat treatments in vitro. This methodology could be applied to the synthesis of other proteins coenzyme dependent, that is PLP dependent.

[0216] Therefore, in the present invention, the conjugation of GOT to PLP can, in a non-limited manner, be carried out in accordance with the new in vivo method for conjugating GOT to PLP illustrated for the first time in the present invention. Such in vivo methodology is exemplified in the present invention, in particular, in the example where we explore the possibility of in vivo conjugation of GOT1 with PLP by co-inoculations of different concentrations of vitamin B6 forms such as PLP, pyridoxine or pyridoxal, jointly with the recombinant baculovirus expressing the construct GOT1-GS4-Hinge-Fc N297G. The influence of PLP on expression of GOT1 and the activity of the enzyme in the soluble fraction of the pupae extracts were then analyzed.

[0217] The tested concentrations of PLP in the co-inoculation were 0 mM, 50 mM, 75 mM and 100 mM. The PLP (P6280, SigmaAldrich) was dissolved at a concentrated preparation in cell culture medium and added to the baculovirus preparation to achieve the indicated final concentrations, without altering the viral dose per insect pupa.

[0218] To evaluate GOT1 productivity, pupae from each condition were processed and mechanically homogenized using an extraction buffer. An extract was taken from each condition and centrifuged. Protein expression and solubility were checked using SDS-PAGE and Coomassie Blue Gel Staining. As already indicated, co-inoculation with PLP at 50 mM, 75 mM, and 100 mM increased GOT1 activity in vitro by more than four times compared to the extract without PLP (0 mM). Apparently, Ranges between 50 - lOOmM of PLP were the best conditions for increasing GOT1 activity, suggesting the best conjugation of the enzyme and maintaining optimal GOT1 productivities.

[0219] Hence, a further embodiment of the ninth aspect of the invention, refers to a method of producing the recombinant PLP conjugated fusion protein of the invention comprising providing a host expression system, introducing a nucleic acid sequence encoding the recombinant fusionprotein into said host system, cultivating the host under conditions conducive to protein expression, and subsequently preferably isolating and purifying the recombinant fusion protein; wherein the method is carried out in the presence of vitamin B6 forms such as PLP, pyridoxine or pyridoxal, ensuring that the conjugation occurs as the protein is expressed and / or processed; and wherein the host system is infected, transfected, transduced or transformed with a composition comprising i) a recombinant baculovirus, vector, or nucleic acid sequence, comprising a nucleic acid sequence encoding a recombinant protein operably linked to a promoter, wherein the recombinant protein is a PLP dependent enzyme preferably a fusion protein as defined in any of the embodiments, or combinations thereof, of the first aspect of the invention, and ii) PLP (Pyridoxal phosphate) or other forms of vitamin B6 such as Pyridoxal o Pyridoxine, more preferably at a concentration above 50 mM preferably between 50 mM and 200 mM, more preferably between 50 mM and 100 mM. Preferably, the host is selected from the group consisting of bacterial, yeast, fungal, mammalian, plant, and insect cells, or living insect pupae or larvae, based on desired protein yield, quality, and post-translational modifications. Preferably, the host system is an insect cell line, such as Trichoplusia ni or Spodoptera frugiperda cells. Preferably, the host comprises insect pupae of the genus Trichoplusia, Rachiplusia, or Bombyx. Preferably, the host system is a mammalian cell line such as CHO or HEK293 cells. Preferably, the host is a yeast strain, such as Pichia pastoris, engineered to express the recombinant fusion protein of the invention under an inducible promoter. Preferably, the host is a plant expression system (e.g., transient expression in Nicotiana benthamiana leaves).

[0220] In an embodiment, the method comprises the following steps:

[0221] a. infecting, transfecting, transducing or transforming an isolated host with a composition comprising:

[0222] i. a recombinant baculovirus, vector, or nucleic acid sequence, comprising a nucleic acid sequence encoding a recombinant protein operably linked to a promoter, wherein the recombinant protein is a PLP dependent enzyme preferably a fusion protein as defined in any of the embodiments, or combinations thereof, of the first aspect of the invention, and

[0223] ii. a PLP (Pyridoxal phosphate) or other forms of vitamin B6 such as Pyridoxal or Pyridoxine, preferably at a concentration above 50 mM, preferably between 50 mM and 200 mM, more preferably between 50 mM and 100 mM.;b. and extracting and optionally purifying the recombinant protein by conventional means, preferably by combining affinity chromatography with protein A resin with other chromatographic and filtration steps in a process similar to monoclonal antibodies purifications.

[0224] Alternatively, the invention refers to a method for producing a recombinant fusion protein according to the first aspect of the invention, comprising the steps of:

[0225] a. infecting, transfecting, transducing or transforming an isolated host with a composition comprising:

[0226] ill. the nucleic acid sequence and / or the vector and / or the bacmid and / or the baculovirus of the invention, wherein the recombinant protein is as defined in any of the embodiments, or combinations thereof, of the first aspect of the invention, and

[0227] iv. a PLP (Pyridoxal phosphate) or other forms of vitamin B6 such as Pyridoxal or Pyridoxine, preferably at a concentration above 50 mM, preferably between 50 mM and 200 mM, more preferably between 50 mM and 100 mM;

[0228] b. and extracting and optionally purifying the recombinant protein by conventional means, preferably by combining affinity chromatography with protein A resin with other chromatographic and filtration steps in a process similar to monoclonal antibodies purifications.

[0229] A tenth aspect of the invention refers to a fusion protein as defined in the first aspect of the invention or in any of its preferred embodiments expressed, obtained or obtainable by the method of production of the recombinant fusion protein of the invention as described in the second aspect of the invention or in any of its preferred embodiments.

[0230] An eleventh aspect of the invention refers to a PLP conjugated fusion protein as defined in the first aspect of the invention or in any of its preferred embodiments expressed, obtained or obtainable by the method of producing the conjugated fusion protein of the invention as described in the nineth aspect of the invention or in any of its preferred embodiments.

[0231] A further embodiment of the invention, refers to a PLP conjugated fusion protein as defined in the first aspect of the invention or in any of its preferred embodiments expressed, obtained or obtainable by the method of producing the recombinant PLP conjugated fusion protein of the invention comprising the steps of providing a host expression system, introducing a nucleic acidsequence encoding the recombinant fusion protein into said host system, cultivating the host under conditions conducive to protein expression, and subsequently preferably isolating and purifying the recombinant fusion protein; wherein the method is carried out in the presence of PLP, ensuring that the conjugation occurs as the protein is expressed and / or processed; and wherein the host system is infected, transfected, transduced or transformed with a composition comprising i) a recombinant baculovirus, vector, or nucleic acid sequence, comprising a nucleic acid sequence encoding a recombinant protein operably linked to a promoter, wherein the recombinant protein is a PLP dependent enzyme preferably a fusion protein as defined in any of the embodiments, or combinations thereof, of the first aspect of the invention, and ii) PLP (Pyridoxal phosphate) or other forms of vitamin B6 such as Pyridoxal o Pyridoxine, more preferably at a concentration above 50 mM preferably between 50 mM and 200 mM, more preferably between 50 mM and 100 mM. Preferably, the host system is selected from the group consisting of bacterial, yeast, fungal, mammalian, plant, and insect cells, or living insect pupae or larvae, based on desired protein yield, quality, and post-translational modifications. Preferably, the host system is an insect cell line, such as Trichoplusia ni or Spodoptera frugiperda cells. Preferably, the host comprises insect pupae of the genus Trichoplusia, Rachiplusia, or Bombyx. Preferably, the host system is a mammalian cell line such as CHO or HEK293 cells. Preferably, the host is a yeast strain, such as Pichia pastoris, engineered to express the recombinant fusion protein of the invention under an inducible promoter. Preferably, the host is a plant expression system (e.g., transient expression in Nicotiana benthamiana leaves).

[0232] It is noted that the extraction and purification of the recombinant protein is preferably carried out by affinity chromatography techniques used for antibodies, and wherein preferably the recombinant protein is expressed or produced in a pupa, comprising the host cell of the invention, belonging to the order Lepidoptera, preferably to the genus Trichoplusia, Rachiplusia or Bombix mori, even more preferably to the genus Trichoplusia and to the species Trichoplusia ni.

[0233] As used herein, by affinity chromatography, it is herein understood as a biochemical separation technique that utilizes the reversible interaction between specific protein domains or nonprotein groups with a binding partner immobilized on a stationary substrate such as agarose beads. Generally, this process involves a passing a mixture containing the target molecule through a column packed with the stationary phase, washing away the unbound material and eluting the target molecules by changing environmental conditions such us pH, conductivity or presence of a competitor molecule.Pharmaceutical composition of the invention and medical uses

[0234] A twelfth aspect of the invention refers to a pharmaceutical composition comprising the fusion protein as defined in the first aspect of the invention or the protein as defined in the tenth or eleventh aspect of the invention, together with a pharmaceutically acceptable carrier, adjuvant or diluent.

[0235] A thirteenth aspect of the invention refers to the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, for use in therapy.

[0236] A fourteenth aspect of the invention refers to the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, for use in a method of reducing extracellular brain glutamate levels, the method comprising administering to a subject in need thereof an agent capable of modulating stress hormone activity thereby reducing blood glutamate levels, thereby reducing extracellular brain glutamate levels. Preferably, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is for use in a method of treatment of a medical condition selected from the group consisting of brain anoxia, stroke, perinatal brain damage, traumatic brain injury, bacterial meningitis, subarachnoid hemorrhage, hemorrhagic shock, epilepsy, acute liver failure, glaucoma, amyotrophic lateral sclerosis, HLV, dementia, hemorrhagic shock, open heart surgery, aneurism surgery, coronary artery bypass surgery grafting and Alzheimer's disease. More preferably, for use in a method of treatment of brain anoxia or brain stroke, in particular for the treatment of ictus. Interestingly, the timing of administration of the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention is important; in this sense, rGOT administered before reperfusion failed to confer significant protection, whereas delivery at the onset of reperfusion resulted in a significant reduction in lesion volume. These results indicate that restored cerebral blood flow is essential for rGOT to reach the ischemic tissue and exert its neuroprotective effect. The demonstrated safety profile and lack of pharmacological interaction between rGOT and rtPA (or the new thrombolytic variant, TNK) further support the feasibility ofthis combination, advocating for the use of rGOT as a complementary therapy to enhance thrombolytic efficacy when delivered immediately before or during reperfusion in acute ischemic stroke. That is, preferably, in the method of treatment of the fourteenth aspect of the invention, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is administered to a subject in need thereof immediately before, after or during reperfusion in diseases such as acute ischemic stroke, preferably in ictus. Furthermore, as indicated in the examples, the interaction assay between GOT and reperfusion agents (rtPA and TNK) was an important component of this invention. Combination therapy can reveal unforeseen pharmacodynamic antagonisms with reperfusion drugs, as exemplified by the ESCAPE-NA1 trial, in which the promising neuroprotective drug, nerinetide, lost its efficacy in rtPA-treated patients due to plasmin-mediated cleavage of its C-terminal sequence.27, 28 In contrast, GOT-Fc retained its catalytic integrity and functionality in the presence of both rtPA and TNK, confirming its biochemical compatibility and supporting its use as a safe adjunctive therapy to existing reperfusion protocols. Therefore, in a preferred embodiment, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is for use in a method of reducing extracellular brain glutamate levels, preferably in a subject having a medical condition such as diabetes and / or hyperglycemia, wherein the method comprising administering to a subject in need thereof the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, immediately before, during reperfusion or subsequently to the administration of a reperfusion agent, preferably rt-PA, t-PAorTNK.

[0237] In the context of the present invention, reperfusion agents can be selected from the list consisting of:

[0238] • Alteplase (brand name Activase, also known as rt-PA or t-PA) is a naturally occurring human tissue plasminogen activator produced by vascular cells. It is the most used fibrinolytic in the treatment of acute ischemic stroke (AIS), acute myocardial infarction (AMI), and pulmonary embolism.

[0239] • Tenecteplase (brand name TNKase, also known as TNK) is a bioengineered variant of alteplase with modifications that give it a longer half-life and greater fibrin specificity.• Reteplase (brand name Retavase) is a bioengineered, modified form of t-PA, also known as rt-PA. It has a faster onset of action and a longer half-life than alteplase, allowing for administration as two separate intravenous bolus injections 30 minutes apart, without a continuous infusion.

[0240] • Streptokinase (brand name Streptase) was one of the first generation of thrombolytic agents and is derived from Streptococcus bacteria.

[0241] • Urokinase (brand names Abbokinase, Kinlytic) is a direct plasminogen activator originally purified from human urine, though recombinant forms are available. It has been used for deep vein thrombosis, pulmonary embolism, peripheral arterial occlusion, and is commonly used for clearing occluded central venous access devices (catheters).

[0242] • Anistreplase (Anisoylated Plasminogen Streptokinase Activator Complex or APSAC, brand name Eminase) is a complex of streptokinase and plasminogen.

[0243] In a preferred embodiment, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is for use in a method of treatment of a medical condition selected from the group consisting of brain anoxia, stroke, perinatal brain damage, traumatic brain injury, bacterial meningitis, subarachnoid hemorrhage, hemorrhagic shock, epilepsy, acute liver failure, glaucoma, amyotrophic lateral sclerosis, HLV, dementia, hemorrhagic shock, open heart surgery, aneurism surgery, coronary artery bypass surgery grafting and Alzheimer's disease; more preferably, for use in a method of treatment of brain anoxia or brain stroke, in particular for the treatment of ictus; preferably in a subject having or suffering from diabetes and / or hyperglycemia; wherein the method comprising administering to a subject in need thereof the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, simultaneously, previously or subsequently to the administration of a reperfusion agent as defined above.

[0244] As indicated in the examples of the present invention, the inclusion of a hyperglycemic model adds further translational relevance to the present invention invention. Hyperglycemia and diabetes are among the most prevalent comorbidities in stroke patients and are well known to worsen clinical prognosis. Diabetic individuals typically exhibit larger infarcts, greater susceptibility to reperfusion injury, impaired microvascular function, and higher rates of hemorrhagic transformation, all of which contribute to poorer functional recovery and increased mortality26. Consistent with this clinical reality, our STZ-induced diabetic rats displayedsignificantly larger lesions than normoglycemic animals, confirming the deleterious impact of hyperglycemia on ischemic damage. Within this adverse metabolic context, treatment with rGOT produced a modest but consistent trend toward reduced infarct volume at all analyzed time points. Importantly, the administration of rGOT did not exacerbate ischemic injury or worsen functional outcomes under hyperglycemic conditions. This is a non-trivial observation, as many neuroprotective or metabolic agents show reduced efficacy— or even detrimental effects— in diabetic animals, a pattern that often mirrors the poorer response of diabetic patients in clinical trials. Therefore, in a preferred embodiment, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is for use in a method of reducing extracellular brain glutamate levels in a subject having a medical condition such as diabetes and / or hyperglycemia. In a preferred embodiment, the fusion protein as defined in the first aspect of the invention, the protein as defined in the tenth or eleventh aspect of the invention or the pharmaceutical composition as defined in the twelfth aspect of the invention, is for use in a method of treatment of a medical condition selected from the group consisting of brain anoxia, stroke, perinatal brain damage, traumatic brain injury, bacterial meningitis, subarachnoid hemorrhage, hemorrhagic shock, epilepsy, acute liver failure, glaucoma, amyotrophic lateral sclerosis, HLV, dementia, hemorrhagic shock, open heart surgery, aneurism surgery, coronary artery bypass surgery grafting and Alzheimer's disease; more preferably, for use in a method of treatment of brain anoxia or brain stroke, in particular for the treatment of ictus; in a high-risk subject or individual having or suffering from diabetes and / or hyperglycemia.

[0245] The following examples merely illustrate but do not limit the present invention.

[0246] EXAMPLES

[0247] Example 1

[0248] Materials and Methods.

[0249] Proteins design and sequences

[0250] The sequence corresponding to GOT1 was cloned in baculovirus vector to obtain the protein with an His Tag: 6His TEV GOT1 (SEQ ID NO 1) (Figure 1)

[0251] MHHHHHHENL YFQSMAPPSV FAEVPQAQPV LVFKLTADFR EDPDPRKVNL GVGAYRTDDC HPWVLPWKK VEQKIANDNS LNHEYLPILG LAEFRSCASR LALGDDSPAL KEKRVGGVQS LGGTGALRIG ADFLARWYNG TNNKNTPVYV SSPTWENHNA VFSAAGFKDI RSYRYWDAEKRGLDLQGFLN DLENAPEFSI WLHACAHNP TGIDPTPEQW KQIASVMKHR FLFPFFDSAY QGFASGNLER DAWAIRYFVS EGFEFFCAQS FSKNFGLYNE RVGNLTWGK EPESILQVLS QMEKIVRITW SNPPAQGARI VASTLSNPEL FEEWTGNVKT MADRILTMRS ELRARLEALK TPGTWNHITD QIGMFSFTGL NPKQVEYLVN EKHIYLLPSG RINVSGLTTK NLDYVATSIH EAVTKIQ*

[0252] To improve the half-life of the GOT1 in blood a human Fc is added to the sequence. Fc region naturally dimerize but can be maintained monomeric with punctual mutations in its CH3 region. Monomeric mutated Fc maintain the characteristic pH-dependent FcRn binding of IgGl Fc. Two strategies have been used to generate GOTl-Fc fusion proteins:

[0253] • Monomeric Fc union to GOT1 to obtain a molecule small and stable. A set of five CH3 mutations were added to the Fc sequence to obtain the monomer Fc.

[0254] • Dimeric Fc union to GOT1, without CH3 mutations and with a Hinge mutated to improve the fragmentation resistance. In this strategy two length of linkers were chosen GS2 and GS4.

[0255] In both strategies a mutation in position 297 was used, that resulted in lack of glycosylation and thus loss of effector function. (Figure 1)

[0256] The following sequences were used to generate three baculoviruses. GOT1 sequence is in bold and Fes in italics, linkers are underlined.

[0257] G0Tl-GS2-mFc 6Mut (SEQ ID NO 2)

[0258] MAPPSVFAEV PQAQPVLVFK LTADFREDPD PRKVNLGVGA YRTDDCHPWV LPWKKVEQK IANDNSLNHE YLPILGLAEF RSCASRLALG DDSPALKEKR VGGVQSLGGT GALRIGADFL ARWYNGTNNK NTPVYVSSPT WENHNAVFSA AGFKDIRSYR YWDAEKRGLD LQGFLNDLEN APEFSIWLH ACAHNPTGID PTPEQWKQIA SVMKHRFLFP FFDSAYQGFA SGNLERDAWA IRYFVSEGFE FFCAQSFSKN FGLYNERVGN LTWGKEPES ILQVLSQMEK IVRITWSNPP AQGARIVAST LSNPELFEEW TGNVKTMADR ILTMRSELRA RLEALKTPGT WNHITDQIGM FSFTGLNPKQ VEYLVNEKHI YLLPSGRINV SGLTTKNLDY VATSIHEAVT KIQGGGGSGG GGSAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYG STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ LTKNQVSLRC AVEWESNGQP E . NNYKTTKPV LDSDGSFFLY STLTVDKSRW QQGNVFSCSV LHEALHNHYT QKSLSLSPGK*

[0259] GOTl-GS2-Hinge-Fc N297G (SEQ ID NO 3)

[0260] MAPPSVFAEV PQAQPVLVFK LTADFREDPD PRKVNLGVGA YRTDDCHPWV LPWKKVEQK IANDNSLNHE YLPILGLAEF RSCASRLALG DDSPALKEKR VGGVQSLGGT GALRIGADFL ARWYNGTNNK NTPVYVSSPT WENHNAVFSA AGFKDIRSYR YWDAEKRGLD LQGFLNDLEN APEFSIWLH ACAHNPTGID PTPEQWKQIA SVMKHRFLFP FFDSAYQGFA SGNLERDAWA IRYFVSEGFE FFCAQSFSKN FGLYNERVGN LTWGKEPES ILQVLSQMEK IVRITWSNPP AQGARIVAST LSNPELFEEW TGNVKTMADR ILTMRSELRA RLEALKTPGT WNHITDQIGM FSFTGLNPKQ VEYLVNEKHI YLLPSGRINV SGLTTKNLDY VATSIHEAVT KIQGGGGSGG GGSDKTYTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYG STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK*GOTl-GS4-Hinge-Fc N297G (SEQ ID NO 4)

[0261] MAPPSVFAEV PQAQPVLVFK LTADFREDPD PRKVNLGVGA YRTDDCHPWV LPWKKVEQK IANDNSLNHE YLPILGLAEF RSCASRLALG DDSPALKEKR VGGVQSLGGT GALRIGADFL ARWYNGTNNK NTPVYVSSPT WENHNAVFSA AGFKDIRSYR YWDAEKRGLD LQGFLNDLEN APEFSIWLH ACAHNPTGID PTPEQWKQIA SVMKHRFLFP FFDSAYQGFA SGNLERDAWA IRYFVSEGFE FFCAQSFSKN FGLYNERVGN LTWGKEPES ILQVLSQMEK IVRITWSNPP AQGARIVAST LSNPELFEEW TGNVKTMADR ILTMRSELRA RLEALKTPGT WNHITDQIGM FSFTGLNPKQ VEYLVNEKHI YLLPSGRINV SGLTTKNLDY VATSIHEAVT KIQGGGGSGG GGSGGGGSGG GGSDKTYTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYG STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK*

[0262] G0T1 (SEQ ID NO 5)

[0263] MAPPSVFAEV PQAQPVLVFK LTADFREDPD PRKVNLGVGA YRTDDCHPWV LPWKKVEQK IANDNSLNHE YLPILGLAEF RSCASRLALG DDSPALKEKR VGGVQSLGGT GALRIGADFL ARWYNGTNNK NTPVYVSSPT WENHNAVFSA AGFKDIRSYR YWDAEKRGLD LQGFLNDLEN APEFSIWLH ACAHNPTGID PTPEQWKQIA SVMKHRFLFP FFDSAYQGFA SGNLERDAWA IRYFVSEGFE FFCAQSFSKN FGLYNERVGN LTWGKEPES ILQVLSQMEK IVRITWSNPP AQGARIVAST LSNPELFEEW TGNVKTMADR ILTMRSELRA RLEALKTPGT WNHITDQIGM FSFTGLNPKQ VEYLVNEKHI YLLPSGRINV SGLTTKNLDY VATSIHEAVT KIQ

[0264] mFc 6Mut (SEQ ID NO 6)

[0265] APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLRCHVK GFYPSDIAVE WESNGQPENN YKTTKPVLDS DGSFFLYSTL TVDKSRWQQG NVFSCSVLHE ALHNHYTQKS LSLSPGK

[0266] Hinge-Fc N297G (SEQ ID NO 7)

[0267] DKTYTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYGSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

[0268] Baculovirus generation and analysis of recombinant proteins expression

[0269] The gene sequences designed were optimized in their use of codons for expression in insect cells and synthesized to be used to obtain the corresponding recombinant baculoviruses, using the Bac-to-Bac system (Invitrogen), based on the transposition of the sequence of interest from the pFastBac vector to the baculoviral DNA. The donor vectors were used to transform DHIOBac E. coli competent cells which contains a helper plasmid expressing the transposase jointly with a bacmid with the baculovirus genome carrying a prokaryotic replication origin and the mini Tn7-replicon.

[0270] The bacmids generated were isolated and checked by PCR to confirm the correct genes transposition (data shown in passage 1 PCR test). The purified bacmid DNAs were transfected tosf9-RVN cells free of rhabdovirus. The cell supernatant harvested from transfected cells contain the passage 0 of each BV generated (pO). These pO viruses were used as inoculum for an amplification round in the same cell line, generating the corresponding passage 1 viruses (pl). The BVs passages 1 were analyzed by PCR with reactions designed with specific primers to detect either, the whole transposition cassette (PCR 1) or the expression cassette (PCR 2), an empty baculovirus generated without gene transposition was used as negative control. Expected size bands were obtained in both PCRs. (Figure 2)

[0271] As an initial analysis of protein expression, pl BVs were used to infect sf9-RNV cells. Samples of cell infected cultures were collected, spun down and the pellet was lysed with RIPA buffer. The lysates were loaded into SDS-PAGE gels which were Coomassie blue stained and also analyzed by Western Blot. The result of these studies is shown in Figure 3.

[0272] Once BVs for each GOT1 version were produced, optimization of proteins expression were carried out in CrisBio expression system (insect pupae as living biofactories).

[0273] Different BV doses between 5,000-50,000 PFUs / insect were tested using a robotic inoculator (Proydes Automatizacion S.L.). The pupae were then incubated in a climatic chamber (PHC Corporation, Biomedical Division) at temperatures ranging from 23-289C and for periods of 3 to 6 days post-infection. The infected pupae were stored at -209C in a freezer.

[0274] To evaluate productivity, 5g of pupae from each condition were processed and mechanically homogenized using as extraction buffer 80 mL of Tris 20 mM, NaCI 500 mM, PMSF ImM, DTT 5m M, pH 8. One mL of extract was taken from each condition and centrifuged at 13000xg, 15 min and 49C to split sample in soluble and insoluble fractions. Protein expression and solubility were checked using SDS-PAGE and Coomassie blue staining. Band densitometry and relative quantification were performed with ImageLab software analysis.

[0275] Purification of 6His-TEV-GOTl

[0276] The biomass was processed at a 1:16 ratio in the extraction buffer. The insect biomass (62.5 g of frozed pupae) was extracted in 1 L of extraction buffer (20 mM Tris, 500 mM NaCI, 1 mM PMSF, 5 mM DTT, pH 8). The processing was carried out using a vertical colloidal mill (Lleal, S.A.). The cuticle was removed using a 25 pm filter (Sentinel NMO-25-P03Z-50S, Eaton). The extract was then homogenized using a high-pressure homogenizer (Lab homogenizer Panda Plus 2000, GEA) with one cycle and pressure adjusted to 300 bars, keeping the material cold using a refrigerated jacket. The extract was centrifuged at 13.000xg for 30 minutes at 49C (Gyozen 1736R, rotor GRF-L-250-6). The resulting supernatant was filtered through a 0.45 pm nitrocellulose membrane(Merck Millipore Ltd) and then loaded onto 45 ml of IMAC Sepharose 6 Fast Flow (Cytiva Uppsala, Sweden AB) pre-equilibrated with buffer (50 mM Tris, 200 mM NaCI, 30 mM Imidazole, pH 7.5). Next, a wash buffer (50 mM Tris, 200 mM NaCI, 1% Deviron, pH 7.5) was circulated at a slow flow rate (20 minutes residence time) to remove contaminants. The column was re-equilibrated with buffer A and eluted with a 0-100% B gradient over 10 CV (Buffer B: 20 mM Tris, 200 mM NaCI, 500 mM Imidazole, pH 7.5). The entire peak was collected. The chromatography was performed using a chromatograph AKTA Go (GE). The samples were submitted to dialysis using a 3.5 kDa membrane (Standard RC Tubing, Spectra / Por), against dialysis buffer (50 mM Phosphate, 100 mM NaCI, pH 7.5). The dialyzed material was filtered with a 0.2 pm syringe filter (Fuktre syringe, Clear Line) and stored at -209C. The purification process and material purity were monitored using SDS-PAGE gels (Mini-Protean TGX Stain-Free Gels, BioRad). The gels were run at 200V for 38 minutes using a power supply (Power Pac Basic, BioRad), followed by staining with Coomassie Blue (Quick Coomassie Stain, Neo Biotech) (Figure 4).

[0277] Purification of GOTl-GS2-mFc 6Mut, GOTl-GS2-Hinge-Fc N297G and GOTl-GS4-Hinge-Fc N297G

[0278] As in the GOTl-His protein, the insect biomass was processed at a ratio of 1:16. The chimeric GOT1 proteins contained in the insect biomass (15.6 g of pupae) were extracted in 0.25 L of extraction buffer (20 mM Tris, 500 mM NaCI, 1 mM PMSF, 5 mM DTT, pH 8.0). For processing, the biomass was mixed with the buffer in a mixing bag (Lateral Bag Filter 400, Interscience) and homogenized using a blender (Bag Mixer, Interscience). The extract was further homogenized by sonication at 50% amplitude, for 3 cycles of 15 seconds (Bendelin, Sonopuls), while keeping the material cold. The extract was then centrifuged at 13,000xg for 30 minutes at 4°C (Gyozen 1736R, rotor GRF-L-250-6). The resulting supernatant was filtered through a depth filter K700P (Pall Corporation) in a Velapad system with a 90 mm diameter (Pall Corporation), followed by filtration through a 1.2 pm glass microfiber filter (Whatman GF / C). The filtrate was then loaded onto 8 ml of Mab Select resin (Cytiva), pre-equilibrated with buffer A (1 mM Tris, 150 mM NaCI, pH 7.4). A wash buffer (10 mM Phosphate, 0.5% Tween 80, pH 5.8) was circulated to stabilize the pH at 6.0. The elution was performed with a 100% B buffer (100 mM Glycine, 0.5% Tween 80, pH 3.5) over 10 column volumes (VC). The entire elution peak was collected. Chromatography was conducted using an AKTA Go system (GE). The eluate was immediately neutralized by dropwise addition onto 200 mM bis-tris, pH 6.0, with vigorous magnetic stirring to prevent aggregation and precipitation. The samples were submitted to dialysis using a 3.5 kDa membrane (Standard RC Tubing, Spectra / Por), against dialysis buffer (50 mM Sodium Acetate, 0,1% Tween 80, pH 6). Thedialyzed material is filtered with a 0.2 pm syringe filter (Fuktre syringe, Clear Line) and stored at -209C.

[0279] The purification process and material purity were monitored using SDS-PAGE gels (Mini-Protean TGX Stain-Free Gels, BioRad). The gels were run at 200V for 38 minutes using a power supply (Power Pac Basic, BioRad), followed by staining with Coomassie Blue (Quick Coomassie Stain, Neo Biotech) (Figure 4). Additionally, Western blot analysis was performed using a transfer kit (Trans-Blot Turbo RTA Midi 0.2 pm Nitrocellulose Transfer Kit, BioRad). The membrane was incubated with an anti-GOTl monoclonal antibody (GT638, Life Technologies) followed by an HRP-conjugated anti-mouse antibody (NXA931V, Cytiva). Another Western blot was performed with incubation using an anti-Fc-HRP antibody (W4031, Promega). The readings from the gels and membranes were acquired using a transilluminator (ChemiDoc MP Imaging System, BioRad).

[0280] GOT1 proteins activity measurement

[0281] The AST / GOT kit (Biosystems Biotech Spain) was used for the GOT1 proteins activity measurement. GOT catalyzes the transfer of the amino group from aspartate to 2-oxoglutarate, forming oxaloacetate and glutamate. The catalytic concentration is determined using the coupled reaction of malate dehydrogenase (MDH), by measuring the rate of NADH disappearance at 340 nm. The kit provides the necessary calculations to obtain GOT activity in U / L, making it possible to calculate U / mg if the concentration of the studied material is known (Figure 5). This activity measurement kit is used both for monitoring the process (extract, material from each purification step) and in the in vivo assay to evaluate GOT1 activity in the blood over time. For measurements during the purification process, dilutions are made to reach concentrations that allow for a linear reaction within a time range of 4 minutes. A plate reader (BioTek Synergy Hl, Agilent) was used with the temperature set at 379C and agitation of the plate prior to each reading. The measurement was performed in a 96-well plate, with 200 pl of the kit reagent plus 10 pl of the sample dilution.

[0282] / fxQ6 Using the data obtained and applying the following formula, the AA / min x - = U / Lf•*. • activity in U / L was determined, and from this, the activity in s x I x VS

[0283] U / mg could be calculated.

[0284] Animal stroke model for GOT and analysis of neuroprotective effect of GOTl-Fc protein Protective effect of GOTl-Fc protein was tested in a transient intraluminal middle cerebral artery occlusion (tMCAO) rat model (Male Sprague-Dawley rats). Transient focal ischemia (60 min) was induced by intraluminal MCA occlusion as previously described17-29using commercially availablesutures with silicone rubber-coated heads (350 pm in diameter and 1.5 mm long; Doccol, Sharon, MA, USA). Cerebral blood flow was monitored with a Periflux 5000 laser Doppler perfusion monitor (Perimed AB, Jarfalla, Sweden) by placing the Doppler probe (model 411; Perimed AB) under the temporal muscle at the parietal bone surface near the sagittal crest. Once artery occlusion was achieved, as indicated by Doppler signal reduction, each animal was carefully moved from the surgical bench to the MR system for baseline ischemic lesion assessment using MRI apparent diffusion coefficient (ADC) maps (before treatment administration). MR angiography (MRA) was also performed to ensure that the artery remained occluded throughout the procedure and to detect possible arterial malformations.30After basal MR analysis, the animals were returned to the surgical bench and the Doppler probe was repositioned. Reperfusion was performed 60 min after the onset of occlusion. In line with our previous study using the same ischemic model, the following exclusion criteria were used:29(1) <70% reduction in the relative cerebral blood flow during arterial occlusion, (2) arterial malformations, as determined by MRA, (3) baseline lesion volume measured using ADC maps, (4) absence of reperfusion or prolonged reperfusion (>10 min until achieving >50% of the baseline cerebral blood flow) after filament removal, and (5) failure to complete treatment. MRI-T2 scans for infarct assessment were performed at 1, 7, and 14 days after ischemia.

[0285] Treatment administration and collection of blood in rats: treatment administration was performed through the i.v. route through the tail vein, and each tested dose was adjusted to a final volume of 1 mL. Blood samples for GOT and glutamate analyses were collected from the tail vein into test tubes (BD Microtainer K2E Tubes, Franklin Lakes, New Jersey, USA).

[0286] Furthermore, the protective effect of the selected dimeric form GOTl-GS4-Hinge-Fc N297G was tested in the transient intraluminal middle cerebral artery occlusion (tMCAO) rat model. Treatment with the chimeric GOTl-Fc protein (GS4 form, 1 mg / kg) was evaluated under two experimental conditions; before arterial recanalization (45 min after artery occlusion) and immediately after arterial recanalization (after 60 min of arterial occlusion). Analysis of blood GOT activity revealed a marked increase relative to basal levels (70 ± 10 U / L), reaching peak values of ~3,500 U / L at 2 h. Activity remained above 1,000 U / L for at least 24 h and returned to baseline by day 14 post-injection. Increase of GOT blood profile was observed in both rGOT-Fc administered before reperfusion (rGOT_preR) and after reperfusion (rGOT_postR). GOT activity in untreated ischemic animals (control group) remained stable during the follow-up period of the study.Animals treated with rGOT_postR showed a progressive and significant reduction in infarct volume compared with both control and pre-reperfusion (rGOT_preR) groups. Specifically, infarct size decreased from 30.8 ± 2.9 mm3in controls to 22.9 ± 2.6 mm3in rGOT_postR at 1 day, from 23.6 ± 2.1 mm3to 16.8 ± 1.7 mm3at 7 days, and from 22.2 ± 1.8 mm3to 16.2 ± 1.6 mm3at 14 days (p < 0.05). In contrast, the rGOT_preR group did not exhibit significant protection relative to controls, maintaining infarct volumes of 31.1 ± 3.0 mm3, 24.4 ± 2.3 mm3, and 23.1 ± 2.1 mm3at 1, 7, and 14 days, respectively. These findings indicate that the therapeutic efficacy of rGOT is maximized when administered immediately after reperfusion. Baseline lesion assessment (day 0) performed during MCA occlusion confirmed consistent ischemic injury across all groups prior to treatment administration, with infarct volumes of 41.9 ± 2.6 mm3in controls, 42.5 ± 2.8 mm3in rGOT_preR, and 40.9 ± 2.5 mm3in rGOT_postR (no significant differences, p > 0.05). Infarct size was calculated as the percentage of ischemic tissue relative to the ipsilateral hemisphere volume and corrected for edema.

[0287] Functional outcome analysis revealed that GOT treatment did not significantly modify motor recovery after ischemia. Both grip strength and laterality index showed comparable values across control, rGOT_preR, and rGOT_postR groups at 7 and 14 days. All groups followed a similar temporal trajectory, characterized by a modest improvement at 14 days likely reflecting spontaneous recovery rather than a treatment effect.

[0288] In vitro functionality of GOT1 proteins produced in insects

[0289] The functionality of the different GOT1 constructs fused to Fc were compared to the native tagged GOT1 protein. The activity units were expressed relative to the concentration of each construct (quantified by anti-GOT western blot), resulting in U / mg. All proteins, fused or not to Fc showed similar activities per mg in the in vitro assay, concluding that the fusion with the constant immunoglobulin region did not affect the GOT1 activity (Figure 5).

[0290] In vivo conjugation of GOT1 with PLP

[0291] The conjugation of GOT1 with PLP may lead to an increase in enzymatic activity, as it is an important coenzyme. We explore the possibility of in vivo conjugation of GOT1 with PLP by coinoculations of different concentrations of PLP jointly with the recombinant baculovirus expressing the construct GOTl-GS4-Hinge-Fc N297G. The influence of PLP on expression of GOT1 and the activity of the enzyme in the soluble fraction of the pupae extracts were then analyzed.The tested concentrations of PLP in the co-inoculation were 0 mM, 50 mM, 75 mM, 100 mM, and 200 mM. The PLP (P6280, SigmaAldrich) was dissolved at a concentrated preparation in cell culture medium and added to the baculovirus preparation to achieve the indicated final concentrations, without altering the viral dose per insect pupa.

[0292] To evaluate GOT1 productivity, 5g of pupae from each condition were processed and mechanically homogenized using as extraction buffer 80 mL of Tris 20 mM, NaCI 500 mM, PMSF ImM, DTT 5m M, pH 8. One mL of extract was taken from each condition and centrifuged at 13000xg 15 min and 49C to split sample in soluble and insoluble fractions. Protein expression and solubility were checked using SDS-PAGE and SYPRO* Ruby Protein Gel Staining. Geles were submitted to bands densitometry analysis and relative quantification with ImageLab software. Additionally, the GOT1 activity in the different extracts was also analyzed.

[0293] Co-inoculation with PLP at 50 mM, 75 mM, and 100 mM increased GOT1 activity in vitro by more than four times compared to the extract without PLP (0 mM). Apparently, 50-100mM were the best conditions for increasing GOT1 activity (Figure 6A), suggesting the best conjugation of the enzyme and maintaining optimal GOT1 productivities (Figure 6B).

[0294] Once determined the optimal PLP concentration for co-inoculations, the other GOT1 constructs were produced in the same conditions and analyzed using Western blot and activity tests. Figure 7 shows that the in vivo GOT1-PLP conjugation was achieved in all recombinant GOTl-derived proteins analyzed.

[0295] In vivo pharmacokinetic analysis of Native and Chimeric GOT1 proteins conjugated with PLP Pharmacokinetic analysis of the chimeric GOTl-Fc protein was tested in healthy experimental animals to confirm the in vivo functionality of the enzyme. The analysis was performed in male rats (n=3) treated with the different GOTlproteins conjugated in pupa (75mM PLP).

[0296] Treatment administration and collection of blood in rats:

[0297] Treatment administration was performed through the i.v. route through the tail vein, and each tested dose was adjusted to a final volume of 1 mL. Blood samples for GOT and glutamate analyses were collected from the tail vein into test tubes (BD Microtainer K2E Tubes, Franklin Lakes, New Jersey, USA). The protein doses were of lmg / Kg of weight to determine the half-life of the proteins in blood. With this experiment we wanted to demonstrate the half-life of the insect-derived GOT1 in blood and how it compares with a control native GOT1 conjugated in vitro chemically. The GOT1 activity in blood was measured at time points of 1, 2, 4, 6, 24, 48, and 72 hours post injection. The level of GOT1 in blood at lh post-injection was considered as 100% ofGOT1 activity. Results clearly showed that the fusion of the Fc immunoglobulin fragment to the GOT1 protein improved the pharmacokinetic of the enzyme, extending its half-life. While the native GOT1 activity in blood was reduced to the 50% in less than 4h, the chimeric GOTl-Fc proteins showed a half-life up to 48h post inoculation. The GOT1 activities of chimeric forms conjugated in vivo and native GOT1 conjugated in vitro were very similar at lh after injection. This suggest that in vitro and in vivo conjugations rendered similar GOT1 activities, despite the differences in complexity or losses of the recombinant protein along the different processes. In vivo PLP conjugation to GOT1 didn't affect the half-life of the protein in blood. Differences in halflife are due to the incorporation transcriptionally of a Fc immunoglobulin fragment to the enzyme (Figure 8).

[0298] Analysis of the neuroprotective effect of GOTl-Fc chimeric proteins in rats submitted to an experimental ischemic stroke.

[0299] Treatment with GOTl-Fc protein (GS4 chimeric protein GOT1 form) at a dose of 1 mg / kg, administered 60 minutes after ischemic induction, caused a significant increase in blood GOT activity relative to basal levels (70 ± 10 U / L), with peak activities of 3,500 U / L at 2 hours (Figure 9). Blood GOT activity was maintained above 1,000 U / L for at least 24 hours after administration and returned to basal levels by day 14 (post-dose). Assessment of the ischemic lesion (Figure 10) showed a significant reduction of infarct size and protective effect (p<0.05) in the GOT1 treated group (GS4 chimeric protein GOT1 form) compared with the control (treated with saline or vehicle) at 24 hours (31,6 vs 19.7%), 7 days (22,4 vs 13.2%) and 14 days (20,1 vs 11.8%). Infarct size was defined such as percentage (%) of ischemic damage with respect to the ipsilateral hemisphere volume, corrected for brain edema. Basal lesion assessment at day 0 and before treatment administration confirmed the ischemic lesion in all the included animals.

[0300] Evaluation of rGOT treatment in a hyperglycemic model of cerebral ischemia

[0301] Diabetes represents one of the most frequent comorbidities in stroke patients and is known to exacerbate ischemic injury and limit therapeutic efficacy.31 Incorporating this condition allows assessment of the neuroprotective potential of GOT under clinically relevant metabolic stress, thereby providing a more realistic framework to evaluate its robustness and translational potential.

[0302] To evaluate whether systemic hyperglycemia affects the neuroprotective efficacy of GOT, a streptozotocin (STZ)-induced diabetic model was combined with transient middle cerebral artery occlusion (tMCAO)32 (Figure 11). Hyperglycemia was successfully induced in all animals following intravenous STZ administration (60 mg / kg), as evidenced by a persistent increase inblood glucose levels above 350 mg / dL from day 9 onward (Figure 11B). Both groups (Control_HG and rGOT_HG) groups exhibited comparable glucose profiles prior to ischemia, confirming a similar metabolic status before surgery. A transient drop in blood glucose levels was observed after ischemia, likely reflecting reduced food intake during the immediate postoperative period. Administration of recombinant GOT (1 mg / kg) immediately after reperfusion led to a sharp rise in plasma GOT activity, peaking between 2 and 5 h ("'4,000 U / L), and gradually returning to basal levels within 7-14 days (Figure 11C). This pharmacokinetic profile confirms that a robust systemic increase in blood GOT activity is achieved even under hyperglycemic conditions, comparable to that observed in normoglycemic animals. Magnetic resonance imaging (MRI) revealed that hyperglycemia exacerbated ischemic damage compared with previous experiments under normoglycemic conditions (40.9 ± 2.9 vs 30.8 ± 2.9 mm3at 1 day, respectively) (Figure 11D). Quantitative analysis showed that rGOT-treated hyperglycemic animals exhibited a consistent trend toward lower infarct volumes compared with controls at all time points, although these differences did not reach statistical significance. Infarct volumes were 40.9 ± 2.9 vs. 38.9 ± 3.0 mm3at 1 day, 38.6 ± 3.2 vs. 32.7 ± 2.8 mm3at 7 days, and 32.7 ± 2.7 vs. 28.8 ± 2.9 mm3at 14 days for ControIHG and rGOTHG groups, respectively (Figure HE). Behavioral evaluation supported the imaging results. Both the grip strength and cylinder (laterality) tests (Figure 11F-G) showed similar motor and sensorimotor performance between control and rGOT-treated hyperglycemic animals. All groups followed a comparable trajectory, with moderate deficits at 7 days and partial spontaneous recovery at 14 days.

[0303] Interaction Between rGOT and Thrombolytic Agents (rtPA and TNK)

[0304] Intravenous thrombolysis with Alteplase@ (recombinant tissue plasminogen activator, rtPA) and more recently the variant Tenecteplase@ (TNK) are currently the only approved drug for acute ischemic stroke. Considering that participants who received rtPA or TNK could be included in a future clinical trial with rGOT for acute stroke, an interaction study between rGOT and both thrombolytic agents was performed. Using a regulatory standardized clot-lysis assay, pooled plasma samples from acute ischemic stroke patients (non-tPA-treated) and commercial control plasma were tested under co-incubation and post-incubation with rtPA or TNK in the presence or absence of rGOT. Optical density measurements were continuously recorded to determine key parameters describing the clot lysis phase (lysis rate, lysis time, clot area). Tranexamic acid (TXA) was included as a positive antifi brinolytic control to validate the sensitivity of the assay. In pooled plasma from stroke patients, co-administration of rGOT with rtPA or TNK did not produce any significant change in the kinetics of clot dissolution compared to vehicle-treatedconditions. Specifically, lysis rate, lysis time and clot area remained statistically indistinguishable from vehicle samples across all tested concentrations of rGOT, confirming that it does not interact with the fibrinolytic machinery under physiological or pathological plasma conditions (Figure 12).

[0305] Enzymatic activity of rGOT (0.016 and 0.04 pg / ml) following incubation with different thrombolytic agents, including rtPA (0.016, 0.16, 1.6 mg / ml) and TNK (0.004, 0.04, 0.04 mg / ml) remained stable and comparable to the control group, with no evidence of reduced catalytic performance or inhibitory interaction, indicating that thrombolytic drugs do not interfere with the rGOT enzymatic function (Figure 14).

[0306] Materials and methods

[0307] Magnetic Resonance Imaging

[0308] Magnetic resonance imaging (MRI) studies were performed using a 9.4T horizontal bore magnet (Bruker BioSpin, Ettligen, Germany) with 12-cm wide actively shielded gradient coils (440 mT / m). Radiofrequency (RF) transmission was achieved with a birdcage volume resonator; signal was detected using a 4-element arrayed surface coil, positioned over the head of the animal. The head of the animal was fixed with a tooth bar, earplugs, and adhesive tape. Transmission and reception coils were actively decoupled from each other. Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore. MR images were analyse using the ImagJ software (htps: / / imagej.nih.gov / ij / ). And lesion sizes were evaluated using apparent diffusion coefficient (ADC) maps (during occlusion and previous to treatment) and T2 relaxation maps (at 1, 7 and 14 days after ischemia) by manually selecting areas with reduced ADC values or hyper intense T2 values by a blinded researcher. Infarct size was defined as the percentage of the ipsilateral hemisphere affected by ischaemic damage and corrected by oedema. Oedema was measured using midline deviation (MD), which was defined as the ratio between ipsilateral and contralateral hemispheres. In summary, infarct lesion was calculated as follows: (infarct volume [mm3 / MD] / ipsilateral hemispheric area [mm3]) x 100.

[0309] MRI ADC maps

[0310] ADC maps were acquired during MCA occlusion (40 minutes after the onset of ischaemia) using a spin-echo echo-planar imaging sequence with the following acquisition parameters: echo time (ET)= 26.91 ms, repetition time (RT)= 4 s, spectral bandwidth (SW) 200 KHz, 7 b-values of 0, 300, 600, 900, 1200, 1600, and 2000 s / mm2, flip angle (FA)= 909, number of averages (NA)= 4, 14consecutive slices of 1 mm, 24x16 mm2field of view (FOV) (with saturation bands to suppress signal outside this FOV), a matrix size of 96x64 (isotropic in-plane resolution of 250 mm / pixel x 250 mm / pixel) and implemented with fat suppression option. Based on previous studies, the values of ADC in the healthy rat brain normally do not fall below 0.55xl03mm2 / s; Therefore, this threshold provides a convenient means of segmenting abnormal tissue (Neurology. 1995 Jan;45(l):172-7.)

[0311] MR angiography

[0312] tMCAO status was evaluated in a non-invasive manner with the time-of-flight magnetic resonance angiography (TOF-MRA). TOF-MRA scan was performed with a 3D-Flash sequence with the following parameters: ET= 2.5 ms, RT=15 ms, FA= 209, NA= 2, SW= 98 KHz, 1 slice of 14 mm, 30.72x30.72x14 mm3FOV (with saturation bands to suppress signal outside this FOV), a matrix size of 256x256x58 (resolution of 120 mm / pixel x 120 mm / pixel x 241 mm / pixel) and implemented without fat suppression option.

[0313] MRI T2 maps

[0314] Ischaemic lesions were determined from T2-maps calculated from T2-weighted images acquired 1, 7 and 14 days after the onset of ischemia using a Multi Slice Multi Echo (MSME) sequence with: ET= 9 ms, RT= 3 seconds, 16 echoes with 9 ms echo spacing, FA= 1809, NA= 2, SW= 75 KHz, 14 slices of 1 mm, 19.2x19.2 mm2 FOV (with saturation bands to suppress signal outside this FOV), a matrix size of 192x192 (isotropic in-plane resolution of 100 mm / pixel x 100 mm / pixel) and implemented without fat suppression option.

[0315] Hyperglycemia model

[0316] Induction of hyperglycaemia was performed using streptozotocin (STZ) as described elsewhere (Drug Chem Toxicol. 2020 Mar;43(2):165-168. EXCLI J. 2023 Feb 21:22:274-294.). In brief, STZ (S0130, Sigma-Aldrich, St. Louis, MO, USA) freshly prepared using cold HBSS (14175095, Gibco, Germany) buffer and protected from light to prevent degradation. One week before ischemia induction, animals were treated with STZ intravenously (i.v.) through the tail vein at a dose of 60 mg / kg / dayley,. Blood glucose levels were measured using a glucometer (Accu-Chek Guide Me, Accu-Chek, Basel, Switzerland) and glucose test strips (07453736, Accu-Chek, Basel, Switzerland) by puncturing the tail vein and extracting a drop of blood. Normal glucose levels ranged between 90-120 mg / dL. Animals were considered hyperglycaemic when glucose levels were higher than 250 mg / dL 6 days after injection.Sensorimotor tests

[0317] Functional outcome was evaluated using the cylinder test and grip strength test, as previously described for GOT testing (iScience. 2024 Oct 9;27(ll):111108). All assessments were performed by a blinded researcher during the animals' dark cycle under consistent environmental conditions. Tests were conducted 1 day before ischemia (baseline) and at 7- and 14-days postsurgery. Animals displaying pre-existing sensorimotor deficits prior to ischemia were excluded. Grip strength test: Forelimb motor function was assessed using a grip strength device (Bioseb, Pinellas Park, USA). The forelimbs, which are most affected by ischemia, were placed on a metal grid. Animals were allowed to grasp the grid, and were then gently pulled backward by the tail until they released their grip. The procedure was repeated three times at each assessment point, and the maximum force recorded before release was measured. Results are expressed as the mean of the three trials.

[0318] Cylinder test: Forelimb use asymmetry during exploratory behavior was evaluated using the cylinder test. Animals were placed individually in a transparent methacrylate cylinder (20 cm diameter x 40 cm height) positioned on a clear surface, and their behavior was video-recorded for 5 minutes using a camera placed underneath. Recordings were analyzed with VirtualDub software (https: / / www.virtualdub.org /

[0319] Clot Lysis Assay (CLA)

[0320] This assay was based on a turbidimetric assay published by Carter et al. (Arterioscler Thromb Vase Biol. 2007 Dec;27(12):2783-9), with some modifications (Proc Soc Exp Biol Med. 1987 Jan;184(l):98-101), and was conducted to assess the influence of rGOT on the ability of recombinant tissue plasminogen activator (rtPA, Actilyse*) or Tenecteplase (TNK, Metalyse*) to lyse clot formation under thrombolytic conditions. Human plasma samples stored at -80gC at the Neurovascular Research Laboratory (VHIR) facilities from previous studies were used (CEIm protocol 412-2025). Selected patients with samples obtained within the first 6 hours of ischemic stroke onset who did not receive any thrombolytic treatment, and which were not under anticoagulant treatment were selected to create two different pool stroke plasma sample. Pool stroke 1 consisted in n=24 smples (50%females, mean age 78 and NIHSS on admission was 7.9) and pool stroke 2 consisted in n=10 samples (50%females, mean age 76.5 and NIHSS on admission was 7.5. Commercial sodium citrate pooled human plasma (blood derived) (IPLAWBNAC, Innovative Research) was used as a control subjects' sample.

[0321] Citrate plasma samples from stroke patients conserved at -80gC were thawed and 25 pL of each were added to a transparent 96-well immuno plate. To activate the plasma coagulation, 50 pL ofActivation Mix containing 7.5 mM CaCL and 0.03 U / mL human Thrombin (T7009, Sigma) was added to the plate. To induce the lysis of the clot, rtPA (Actylise®, Boehringer, 83 ng / mL) or TNK (184 ng / mL) were added in 75 pL of Assay Buffer containing 0.05 M Tris-HCI (Sigma-Aldrich, USA), 0.1 M NaCI (Sigma-Aldrich) at pH = 7.4, these doses show similar clot lysis profiles in the stroke plasma pool. For drug co-treatment with thrombolytics, addition of rGOT (concentration range from 9.2 to 920 ng / mL), positive control for antifibronolysis (Tranexamic acid, 1107.7 ng / mL, 857653 Sigma-Aldrich), or vehicle (NaCI 0.9% B. Braun) solution were mixed in the 75 pL of Assay Buffer. For drug treatment post-thrombolytics, 10 pL of Assay Buffer were removed at the beginning and mixed with rGOT, positive control, or vehicle solution and added after the assay was initiated (between 15-20 minutes). Optical Densities (OD) were read at 405nm every 40 seconds for 4 hours at 37gC using a BIO-TEK SynergyMx microplate reader and the Gen5 (3.14) imager software (Agilent).

[0322] For each experiment, the differences in pipetting times between columns (10 s) were considered during data analysis. All samples were run in triplicate, and OD values were accepted if the coefficient of variation (CV) was less than 35%. Each independent experiment included two sets of triplicates of inter-assay control showing a CV less than 25 % across all plates. Final number of independent assays per tested condition was n=3-5.

[0323] The changes registered in OD (given in au) through time relate to phases of clot formation and lysis, which generate a turbidimetric curve corresponding to the 9 analyzed parameters of clot formation (1-3) and lysis (4-9) as shown in Figure 12 (based on Carter et al. 2007, with some modifications): 1. Latency time (Lag Time): time until a sharp rise in OD, consistent with the time needed for protofibrils to aggregate and cause a clot to form (given in minutes); 2. Clot formation Rate (CR): maximum change in the OD of clot formation, reflecting the maximum speed of fibrin fiber assembly, (given in au per seconds); 3. Maximum Absorbance (MaxAbs): difference between the maximum OD (OD2) and the baseline OD (OD1), represents the structural density of the thrombus, (given in au); 4. Lysis Rate (LR): maximum change in the OD of fibrin fibers disassembly (given in au per seconds); 5. Lysis Time: duration of fibrinolysis from OD2 to return to baseline OD (OD3) (given in minutes); 6. Clot Area (AUC): area under the curve, the balance between clot formation and lysis; 7. Clot Time: total clot lifetime (given in minutes); 8. Lysis 50 from time 0 (Lys 5Oto): time to reach 50% clot lysis from OD1 (given in minutes); 9. Lysis 50 from time of Maximum Absorbance (Lys 50MA): time to reach 50% clot lysis from OD2 (given in minutes).Quantification and statistical analysis

[0324] All data are expressed as mean ± standard error of the mean. The data were analyzed using SPSS statistical software (vl9.0) and GraphPad Prism software (v.8.3.0) for representation of graphs. BioRender (https: / / biorender.com / ) was used for creating the figures. The criterion for statistical significance was set at p < 0.05. The Shapiro-Wilk test was used to determine whether the data were normally distributed. Based on the results of normality tests and the sample size, statistical analysis was performed using non-parametric tests, Wilcoxon test for paired data, and Mann-Whitney test for unpaired data.

[0325] All data collection and analyses were performed by experimenters who were blinded to the animal's identity and experimental conditions. The exact "n" in each group is specified in the figures. Sample sizes were based on the variance of data in pilot experiments and were generally estimated by power calculations that determined the number of animals required for 80% power to detect a 20-30% difference between groups.References

[0326] 1. Rej R. Measurement of aspartate aminotransferase activity: effects of oxamate. Clinical chemistry. 1979;25:555-9.

[0327] 2. Rej R. Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues. Clinical chemistry. 1978;24:1971-9.

[0328] 3. Abrego J, Gunda V, Vernucci E, Shukla SK, King RJ, Dasgupta A, Goode G, Murthy D, Yu F and Singh PK. GOTl-mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells. Cancer letters. 2017;400:37-46.

[0329] 4. Vijay Kumar KDG. 7b Determine Alanine and Aspartate Transaminase Activity in Serum: Springer, Singapore; 2018.

[0330] 5. Zhang D, Xiao M, Wang L and Jia W. Blood-Based Glutamate Scavengers Reverse Traumatic Brain Injury-Induced Synaptic Plasticity Disruption by Decreasing Glutamate Level in Hippocampus Interstitial Fluid, but Not Cerebral Spinal Fluid, In Vivo. Neurotoxicity research.

[0331] 2019;35:360-372.

[0332] 6. Gruenbaum BF, Kutz R, Zlotnik A and Boyko M. Blood glutamate scavenging as a novel glutamate-based therapeutic approach for post-stroke depression. Therapeutic advances in psychopharmacology. 2020; 10:2045125320903951.

[0333] 7. Ruban A, Berkutzki T, Cooper I, Mohar B and Teichberg VI. Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas. Investigational new drugs.

[0334] 2012;30:2226-35.

[0335] 8. Zhang D, Mably AJ, Walsh DM and Rowan MJ. Peripheral Interventions Enhancing Brain Glutamate Homeostasis Relieve Amyloid beta- and TNFalpha- Mediated Synaptic Plasticity Disruption in the Rat Hippocampus. Cerebral cortex. 2017;27:3724-3735.

[0336] 9. Ruban A, Malina KC, Cooper I, Graubardt N, Babakin L, Jona G and Teichberg VI. Combined Treatment of an Amyotrophic Lateral Sclerosis Rat Model with Recombinant GOT1 and Oxaloacetic Acid: A Novel Neuroprotective Treatment. Neuro-degenerative diseases.

[0337] 2015;15:233-42.

[0338] 10. Castillo J, Loza Ml, Mirelman D, Brea J, Blanco M, Sobrino T and Campos F. A novel mechanism of neuroprotection: Blood glutamate grabber. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[0339] 2016;36:292-301.

[0340] 11. Khanna S, Briggs Z and Rink C. Inducible glutamate oxaloacetate transaminase as a therapeutic target against ischemic stroke. Antioxidants & redox signaling. 2015;22:175-86.12. Boyko M, Stepensky D, Gruenbaum BF, Gruenbaum SE, Melamed I, Ohayon S, Glazer M, Shapira Y and Zlotnik A. Pharmacokinetics of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase and their blood glutamate-lowering activity in naive rats. Neurochemical research. 2012;37:2198-205.

[0341] 13. Boyko M, Gruenbaum SE, Gruenbaum BF, Shapira Y and Zlotnik A. Brain to blood glutamate scavenging as a novel therapeutic modality: a review. Journal of neural transmission.

[0342] 2014;121:971-9.

[0343] 14. Rink C, Gnyawali S, Peterson L and Khanna S. Oxygen-inducible glutamate oxaloacetate transaminase as protective switch transforming neurotoxic glutamate to metabolic fuel during acute ischemic stroke. Antioxidants & redox signaling. 2011;14:1777-85.

[0344] 15. Rink C, Gnyawali S, Stewart R, Teplitsky S, Harris H, Roy S, Sen CK and Khanna S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2017;31:1709-1718.

[0345] 16. Xu J, Khoury N, Jackson CW, Escobar I, Stegelmann SD, Dave KR and Perez-Pinzon MA. Ischemic Neuroprotectant PKCepsilon Restores Mitochondrial Glutamate Oxaloacetate Transaminase in the Neuronal NADH Shuttle after Ischemic Injury. Translational stroke research.

[0346] 2020;11:418-432.

[0347] 17. Perez-Mato M, Ramos-Cabrer P, Sobrino T, Blanco M, Ruban A, Mirelman D, Menendez P, Castillo J and Campos F. Human recombinant glutamate oxaloacetate transaminase 1 (GOT1) supplemented with oxaloacetate induces a protective effect after cerebral ischemia. Cell death & disease. 2014;5:e992.

[0348] 18. Dopico-Lopez A, Perez-Mato M, da Silva-Candal A, Iglesias-Rey R, Rabinkov A, Bugallo-Casal A, Sobrino T, Mirelman D, Castillo J and Campos F. Inhibition of endogenous blood glutamate oxaloacetate transaminase enhances the ischemic damage. Translational research : the journal of laboratory and clinical medicine. 2021;230:68-81.

[0349] 19. Campos F, Rodriguez-Yanez M, Castellanos M, Arias S, Perez-Mato M, Sobrino T, Blanco M, Serena J and Castillo J. Blood levels of glutamate oxaloacetate transaminase are more strongly associated with good outcome in acute ischaemic stroke than glutamate pyruvate transaminase levels. Clinical science. 2011;121:11-7.

[0350] 20. Campos F, Sobrino T, Ramos-Cabrer P, Castellanos M, Blanco M, Rodriguez-Yanez M, Serena J, Leira R and Castillo J. High blood glutamate oxaloacetate transaminase levels are associated with good functional outcome in acute ischemic stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2011;31:1387-93.21. da Silva-Candal A, Perez-Diaz A, Santamaria M, Correa-Paz C, Rodriguez-Yanez M, Arda A, Perez-Mato M, Iglesias-Rey R, Brea J, Azuaje J, Sotelo E, Sobrino T, Loza Ml, Castillo J and Campos F. Clinical validation of blood / brain glutamate grabbing in acute ischemic stroke. Ann Neurol. 2018;84:260-273.

[0351] 22. Perez-Mato M, Dopico-Lopez A, Akkoc Y, Lopez-Amoedo S, Correa-Paz C, Candamo-Lourido M, Iglesias-Rey R, Lopez-Arias E, Bugallo-Casal A, da Silva-Candal A, Bravo SB, Chantada-Vazquez MDP, Arias S, Santamaria-Cadavid M, Estany-Gestal A, Zaghmi A, Gauthier MA, Gutierrez-Fernandez M, Martin A, Llop J, Rodriguez C, Almeida A, Migliavacca M, Polo E, Pelaz B, Gozuacik D, El Yamani N, SenGupta T, Runden-Pran E, Vivancos J, Castellanos M, Diez-Tejedor E, Sobrino T, Rabinkov A, Mirelman D, Castillo J and Campos F. Preclinical validation of human recombinant glutamate-oxaloacetate transaminase for the treatment of acute ischemic stroke. iScience. 2024;27:111108.

[0352] 23. Nettleship JE, Assenberg R, Diprose JM, Rahman-Huq N and Owens RJ. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol.

[0353] 2010;172:55-65.

[0354] 24. Gomez-Casado E, Gomez-Sebastian S, Nunez MC, Lasa-Covarrubias R, Martinez-Pulgarin S and Escribano JM. Insect larvae biofactories as a platform for influenza vaccine production. Protein Expr Purif. 2011;79:35-43.

[0355] 25. Smith GE, Summers MD and Fraser MJ. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983;3:2156-65.

[0356] 26. Hafez S, Coucha M, Bruno A, Fagan SC and Ergul A. Hyperglycemia, acute ischemic stroke, and thrombolytic therapy. Translational stroke research. 2014;5:442-453.

[0357] 27. Hill MD, Goyal M, Menon BK, Nogueira RG, McTaggart RA, Demchuk AM, Poppe AY, Buck BH, Field TS, Dowlatshahi D, van Adel BA, Swartz RH, Shah RA, Sauvageau E, Zerna C, Ospel JM, Joshi M, Almekhlafi MA, Ryckborst KJ, Lowerison MW, Heard K, Garman D, Haussen D, Cutting SM, Courts SB, Roy D, Rempel JL, Rohr AC, lancu D, Sahlas DJ, Yu AYX, Devlin TG, Hanel RA, Puetz V, Silver FL, Campbell BCV, Chapot R, Teitelbaum J, Mandzia JL, Kleinig TJ, Turkel-Parrella D, Heck D, Kelly ME, Bharatha A, Bang OY, Jadhav A, Gupta R, Frei DF, Tarpley JW, McDougall CG, Holmin S, Rha JH, Puri AS, Camden MC, Thomalla G, Choe H, Phillips SJ, Schindler JL, Thornton J, Nagel S, Heo JH, Sohn SI, Psychogios MN, Budzik RF, Starkman S, Martin CO, Burns PA, Murphy S, Lopez GA, English J, Tymianski M and Investigators E-N. Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPE-NA1): a multicentre, double-blind, randomised controlled trial. Lancet. 2020;395:878-887.28. Mayor-Nunez D, Ji Z, Sun X, Teves L, Garman JD and Tymianski M. Plasmin-resistant PSD-95 inhibitors resolve effect-modifying drug-drug interactions between alteplase and nerinetide in acute stroke. Sci Transl Med. 2021;13.

[0358] 29. Fernandez-Susavila H, Iglesias-Rey R, Dopico-Lopez A, Perez-Mato M, Sobrino T, Castillo J and Campos F. Inclusion criteria update for the rat intraluminal ischaemic model for preclinical studies. Disease models & mechanisms. 2017;10:1433-1438.

[0359] 30. Vieites-Prado A, Iglesias-Rey R, Fernandez-Susavila H, da Silva-Candal A, Rodriguez-Castro E, Grohn OH, Wellmann S, Sobrino T, Castillo J and Campos F. Protective Effects and Magnetic Resonance Imaging Temperature Mapping of Systemic and Focal Hypothermia in Cerebral Ischemia. Stroke. 2016;47:2386-96.

[0360] 31. Mosenzon O, Cheng AY, Rabinstein AA and Sacco S. Diabetes and Stroke: What Are the Connections? J Stroke. 2023;25:26-38.

[0361] 32. MacDougall NJ and Muir KW. Hyperglycaemia and infarct size in animal models of middle cerebral artery occlusion: systematic review and meta-analysis. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2011;31:807-18.

Claims

63CLAIMS1. A fusion protein comprising at least one glutamate oxaloacetate transaminase covalently bound or linked, optionally through a peptide linker, to a Fc region (fragment crystallizable region), preferably the human IgGl Fc region or a variant thereof.

2. The fusion protein according to claim 1, wherein the glutamate oxaloacetate transaminase is selected from the list consisting of GPT, glutamate oxaloacetate transaminase 1 (GOT1) and glutamate oxaloacetate transaminase 2 (GOT2).

3. The fusion protein according to claim 1, wherein the glutamate oxaloacetate transaminase is glutamate oxaloacetate transaminase 1 (GOT1).

4. The fusion protein according to any one of claims 1 to 3, wherein the fusion protein is in the form of a dimer protein construct, preferably a homodimer, or in the form of a monomeric protein construct consisting of a single polypeptide chain.

5. The fusion protein according to claim 4, wherein the fusion protein is in the form of a homodimer, wherein each of the monomers, or polypeptide chains, of the dimer protein is a fusion protein comprising at least one glutamate oxaloacetate transaminase 1 (GOT1) covalently linked directly or optionally through a peptide linker, to a variant Fc region, preferably a variant of the human IgGl Fc region, having at least an amino acid substitution in position 297 that prevents the Fc region from being glycosylated at this site; wherein the amino acid numbering is according to the EU numbering, and wherein preferably the variant Fc region, preferably a variant of the human IgGl Fc region, is further characterized by comprising a Hinge mutated to improve the fragmentation resistance, preferably the mutated Hinge of SEQ ID NO 9.

6. The fusion protein according to claim 5, wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein having a single amino acid chain comprising SEQ ID NO: 5, or a sequence having a sequence identity of at least 90% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, preferably through a peptide linker, to the N-terminal of SEQ ID NO 7, or a sequence having a sequence identity of at least 90% with SEQ ID NO 7 that results in lack of glycosylation64at position 297 of the Fc region, wherein the amino acid numbering is according to the EU numbering.

7. The fusion protein according to claim 6, wherein the fusion protein is in the form of a dimer protein construct, preferably a homodimer, wherein each of the monomers or polypeptide chains of the dimer protein is a fusion protein having the sequence set forth in SEQ ID NO 3 or SEQ ID NO 4 or a variant thereof having a sequence identity of at least 90% with SEQ ID No 3 or SEQ ID No 4 that results in lack of glycosylation at position 297 of the Fc region, wherein the amino acid numbering is according to the EU numbering.

8. The fusion protein according to claim 7, wherein each of the monomers of the dimer protein is the sequence set forth in SEQ ID NO 4.

9. The fusion protein according to claim 4, wherein the fusion protein is in the form of a monomer protein construct, wherein the fusion protein comprises a single polypeptide chain having at least one glutamate oxaloacetate transaminase 1 (GOT1) covalently linked directly or optionally through a peptide linker, to a variant Fc region, preferably a variant of the human IgGl Fc region, having one or a combination of amino acid substitutions that prevents the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form.

10. The fusion protein according to claim 9, wherein the fusion protein is a monomeric protein having a single amino acid chain comprising: SEQ ID NO: 5, or a sequence having a sequence identity of at least 90% with SEQ ID NO 5, wherein the C-terminal of SEQ ID NO: 5 is covalently linked, optionally through a peptide linker, to the N-terminal of SEQ ID NO 6, or a sequence having a sequence identity of at least 90% with SEQ ID NO 6 that prevents the fusion protein from forming dimers and thus maintains the fusion protein in monomeric form, optionally through a peptide linker.

11. The fusion protein according to claim 10, wherein the fusion protein is in the form of a monomer protein construct having the sequence set forth in SEQ ID No 2.

12. The fusion protein according to anyone of the precedent claims, wherein the fusion protein is conjugated to PLP (Pyridoxal phosphate).6513. The fusion protein according to claim 8, wherein the fusion protein is conjugated to PLP.

14. The fusion protein according to any of the precedent claims, for use in therapy.

15. The fusion protein according to any one of claims 1 to 13, wherein the fusion protein is for use in a method of treatment of brain stroke.

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