High-affinity neutralizing antibody against ricin

Abstract

The invention relates to the field of passive immunotherapies for the prophylaxis and treatment of poisonings caused intentionally or accidentally by ingestion or inhalation of ricin, a toxin from the castor oil plant Ricinus communis. The present invention relates more specifically to an antibody having a high affinity for the A chain of ricin and which allows in vitro neutralization of the toxin as well as short- and long-term protection against this poisoning. The present invention also relates to a murine / human chimeric antibody and a humanized antibody for the prevention or treatment of ricin poisoning.

Description

Title of the invention: High affinity neutralizing antibody directed against ricin Summary of the invention

[0001] The invention lies in the field of passive immunotherapies for the prophylaxis and treatment of poisonings caused intentionally or accidentally by ingestion or inhalation of ricin, a toxin from the castor oil plant Ricinus communis. The present invention relates more specifically to an antibody having a high affinity for the A chain of ricin and which allows in vitro neutralization of the toxin as well as short and long-term protection against this poisoning. The present invention also relates to a murine / human chimeric antibody and a humanized antibody for the prevention or treatment of ricin poisoning. Description of the prior art

[0002] Ricin is a protein synthesis-inhibiting toxin produced by a shrub of the Euphorbiaceae family, the castor oil plant. Ricin is a highly toxic glycoprotein with a molecular weight of approximately 60-65 kDa, which is formed of two polypeptide chains, A and B, linked together by a disulfide bridge. The B chain is a lectin that binds to galactose-containing glycoproteins and glycolipids expressed on the cell surface, facilitating the entry of ricin into the cytosol. The A chain inhibits protein synthesis by irreversibly inactivating the 28S ribosomal subunit. Ricin is toxic by oral, parenteral, and pulmonary routes.Ricin dispersed in powder or aerosol form can cause signs of eye irritation (burning sensation, tearing, more or less severe conjunctivitis) and throat irritation, as well as more or less marked respiratory irritation: cough, dyspnea, pulmonary edema which can lead to acute respiratory distress syndrome, within a period of several minutes to several hours.

[0003] Ricin is now classified as a Category B biological agent by the Centers for Disease Control and Prevention in the United States (CDC ricin webpage: https: / / emergency.cdc.gov / agent / ricin / clinicians / epidemiology.asp) and Category B by the Organisation for the Prohibition of Chemical Weapons (OPCW) (Table 1 OPCW = https: / / www.opcw.org / en / convention-on-prohibition-of-chemical-weapons / annexes / annex-chemicals / table-1).

[0004] In recent years, a resurgence of terrorist attacks using ricin, previously foiled (in the United States in 2013, in France in 2018, in Germany in 2018 and in 2023), has been observed, not to mention cases of intentional poisoning. (suicides) or occasional accidents in children, adults or animals. To date, no effective medical countermeasure has been approved or is marketed for the prevention or treatment of ricin poisoning. Authorities whose role is to ensure the security of the territory and the population must therefore be prepared for the possibility of a ricin attack. In this context of the fight against bioterrorism, having a medical countermeasure, capable of protecting against or reducing the effects of ricin poisoning, quickly administrable and effective is a necessity.

[0005] Furthermore, ricin exists in two different isoforms (called D and E) and can be extracted and purified from different cultivars, containing varying proportions of these two isoforms, and may have slight variations in peptide sequences from one cultivar to another. It is therefore essential that the medical countermeasures produced have the same level of protection, regardless of the original cultivar and the isoform.

[0006] After exposure to ricin, the following antidotes can be used: sugar analogs preventing the binding of ricin to its target or inhibitors of the catalytic subunit such as razidothymidine. Another strategy for the treatment of ricin poisoning is to administer anti-ricin antibodies. However, passive immunotherapy with antibodies would only be effective if broad-spectrum neutralizing anti-ricin antibodies (i.e., recognizing both D and E isoforms, for ricins from different castor bean cultivars) are available.

[0007] The development of new antibodies capable of neutralizing both isoforms of ricin is thus of general interest for the effective prevention and treatment of ricin poisoning.

[0008] Several anti-ricin antibodies have already been described in the literature ([1] -

[22] ). According to the authors of these studies, these antibodies have good in vitro neutralization and in vivo protection capabilities. However, to date, none of these molecules has been tested in the clinical phase: they cannot be used as a treatment after exposure to the toxin. Only vaccines against ricin have been tested in clinical phase I (RiVax, RVEc™). These products are only intended for prophylaxis. In addition, they need to be injected repeatedly to induce protection and several days to weeks before the presumed poisoning, which is incompatible with an emergency situation.

[0009] The anti-ricin antibodies known to date have different structures and have been obtained by different methods. Some are monoclonal (an antibody of a single sequence, produced by a single cell clone, recognizing only one epitope of the antigen (here ricin)) and others are polyclonal (a mixture of several antibody sequences, produced by different cell clones, and recognizing several epitopes of ricin). These polyclonal antibodies with protective activity against ricin poisoning can be derived from immunizations of animals, most often horses or goats. For this, repeated administrations of the target (either one of the two ricin chains, or the whole inactivated ricin (toxoid)) are carried out, in the presence of adjuvants. The plasma of the animals is then prepared from blood samples, then the antibodies are purified, and when used for therapeutic purposes, are treated enzymatically to remove the constant Fc parts and thus obtain a form of F(ab')2 which is better tolerated by patients. Monoclonal antibodies with protective anti-ricin activity are immunoglobulin G (IgG) of murine origin, or humanized, or nanobodies or VhHs, single-domain antibodies which contain only the variable part of the heavy chain.Another antibody described in the literature, called JJX12, is a bispecific antibody consisting of two anti-ricin VhHs with distinct epitopes, linked by a 15-mer peptide. FR2922212 describes the anti-ricin antibody called “43RCA-G1”.

[0010] The dissociation constants (KD) of the most affinity antibodies published are of the order of nM, when the dissociation is measurable (see Table 1). In general, the isoform and / or cultivar from which the tested ricin originates is not specified, which is a limitation, given the level of specificity of the antibodies.

[0011] [Tables 1] mAbs Kb(nM) {isoform D / ë) Method used cultivar ref PB1O 40(RTA) SPR (Biacore) Not specified [14) RA36 0.35 4 0.046 SPR (Biacore) R. carmencito [1SJ 43 RCA 0.048 + < 0.001 (isoform 0) 0.096 ± < 0.001 (isoform E) BU (Octet) R. carmencita [6} mGD12 2.9 (RTA) SPR (Biacore) Not specified Commercial ricin CAS 9009-86-3 [Wî MHl <0.001 (whole ricin) (no dissociation measured} BU (Octet) Not specified [16J MH 36 < 0.001 (whole ricin) (no dissociation measured} BLI (Octet) Not specified [161 RB34 0.01 + < 0.001 (isotorm D) S.42 ± 0.44 (tsoform E) BU (Octet) R. carmencîta [6] RBÎ7 0.137 ± < 0.001 (isoform Dî Isoform E not recognized BLI (Octet) R.camencito [61 D9 2.55 (ricin, isoform not specified) SPR (SensiQ) Not specified [8] MH2 0.6 (ricin, isoform not specified) BLI (Octet) Not specified [16) MH73 < 0.001 (whole ricin) (no dissociation measured} BU (Octet) Not specified 116} MH75 < 0.001 (whole ricin) (no dissociation measured} BU (Octet) Not specified U6) MH76 3.0 (whole ricin) BLI (Octet) Not specified [16J MH77 0.001 (whole ricin) BU (Octet) Not specified [16) 4C13 0.165 (whole ricin) BLi (Octet) Not specified 117} Goat polyclonal IgG Goat polyclonal F(ab}' 124.5 (whole ricin) 157.1 (whole ricin) SPR (SensiQ) Not specified 118).

[0012] Table 1: affinities of antibodies described in the literature for ricin (source cultivar, isoforms D or E), and method used to measure them (Surface Plasmonic Resonance = SPR and BioLayer Interferometry = BLI).

[0013] These tests consist of placing cells in the presence of ricin (the studies of the prior art do not specify the origin of the ricin or the isoform(s) used), at a certain concentration, with different concentrations of the antibody to be evaluated. Following an incubation time which varies from one test to another, the cell viability is measured. Since the cell line, the incubation time, the ricin cultivar, and the method used to measure the cell viability vary from one test to another, it is not relevant to compare the IC50 (Inhibitory Concentration 50%: concentration of antibody necessary to have 50% cell viability in the presence of ricin) calculated in different tests.

[0014] Furthermore, variation of a single amino acid in the recognition epitope can completely abolish the binding of an antibody. It is therefore essential to precisely characterize the affinities of the antibodies towards the D and E isoforms of ricin, and to know the original cultivar used to determine this affinity.

[0015] Table 2 summarizes the available published data on the in vivo protective performance of the few anti-ricin antibodies described as potentially having a therapeutic effect (i.e. the antibody is administered post-exposure to a certain dose of ricin):

[0016] [Tables2] mAbs mAb urigin Ricin dose (LDSO) Route of intoxication Ab dose Route crf Ab administration Treatment rime pasLexposure Survivai rate atthe end trf assay Assay duration (days) Mtce mode! Ref mRAClB Murine 20 Kg / kg (n.ii.j i.p. 1 nng / kg i.p. 4h 100 y BALB / c [19j P310 Chimeric 16.8 Kg / kg (51DW) aérosol 10 mg / kg i.p. 4h 100 S BALB / c

[20] RA36 Murine 7.5 i.n S mg / kg i,v Ih 60 21 CÙ1 115* 43RCA-G1 Humanized 10O gg / kg i.n. 10 mg / kg iv. 6. 24h 0 21 BALB / c 16] GO12 Chimeric 1Q0 [ig / kg i.p. 100 i;g 200 Kg i.p. 4, 6, &, lOh 100,40,0, 0 100, 80,0. 0 S BALB / c !21i MH1 Chimeric S Kg / kg (20M i.n. 100 pg i. v. 6h 100 14 ICR [16j MH36 Chimeric 5 K8ÀS (2CM i.n. 100 Kg i.v. 6h 95 14 ICH [16ï R334 Murine 100 Kg / kg (5Olso) i.n. 10 mg / kg i.v. 6, 24h "5,0 21 BALB / c ■6] R337 Murine 7.5 Kg / kg (50M i.n. S mg / kg i.v. ih 90 21 CM D9 Murine -lUg (SDlso ) i.p. 5 pg ■I.p. 2, 4, 6h 100, 100,100 7 BALB / c ÎS] MHz Chimeric 5 K8 / kg (2OM' i.n. 100 Kg LV, 611 40 14 ICR [16; MM73 Chimeric 5 pg / kg (2Dlsci i.n 100 Kg t.V 6h SS 14 ICR fl6s MM 75 Chimeric 5 pg / kg Ln. 100 ug i.v. Ch 95 14 ICR iiSî MH76 Chimeric 5|18 / kg(20M' i.n. 100 Rg i.v. 6h 60 14 ICE [16: MM77 Chimeric 5 Kg / kg (2DI.SO1 i.n 100 pg i.v 6h 100 14 ICR

[16] 4C13 Murine 20 Kg / kg (n.d.> I.p. 0.4 mg / kg i.V. 2,6,17, 24n 100. 7S, 0, 0 30 CD1

[17] Polyclonal IgG polyclonal Ftab’h Goat ~1 Kg (SLDso) LP- 3 nmcl i-P- Ih 100 7 BALB / c

[18] Polydona! IgG polyclonal F{ab’h G e st i.n. aucun NA 9 jours après l** exposition et traitement (ligne ci-dessus) 100 o. + 7 C3V5 aftsr 2 exposit ion BALB / c

[18] Polyclonal IgG polyclonal Flab'h Equine 7 Kg / kg (21C5O) Ln. n.d i.n., i.v. 24h 24h,48h 62 {14 deys) 65, 34 (14 deys) 14 CD1 .

[0017] Tableau 2 : performances de protection in vivo des anticorps décrits dans la littérature, i.p : intrapéritonéal ; i.n : intranasal ; i.v : intraveineux ; n.d : non déterminé

[0018] However, it is difficult to compare these results with each other. Indeed, the strains of mice used may be different, the doses of ricin administered may also be different, the equivalences in 50% Lethal Doses (LD50, doses necessary to kill 50% of the mice) and the original cultivar or isoform are not always specified, the routes of administration of ricin and / or the antibody may vary, as well as the quantities of antibodies. Finally, it is common for experiments to be stopped too early, which is favorable in terms of survival rate, when the animals are still in an unstabilized phase and mortality is still observed.

[0019] These anti-ricin monoclonal antibodies allow at best a survival rate greater than 50% in mice. However, they were injected very quickly after exposure to ricin (maximum 6 hours post-exposure) (Table 2), which is not necessarily compatible with field situations (the time between intoxication and access to treatment is often greater than 6 hours, the time for the diagnosis to be made and the patient to be taken care of by a structure available for treatment).

[0020] Only polyclonal F(ab')2 of equine origin allow a survival of 65% 24 hours post-exposure in mice. However, the industrial-scale production of equine polyclonal antibodies has several significant drawbacks compared to the production of monoclonal antibodies (inter-batch reproducibility, expensive immunizations, etc.) and the F(ab')2 format of the antibodies can also raise other problems, particularly bioavailability.

[0021] Thus, it is important today to identify anti-ricin monoclonal antibodies that are easily produced on a large scale, which allow in vitro neutralization of the two isoforms of the toxin as well as strong short- and long-term protection in humans intentionally or accidentally poisoned with this toxin, or prophylactically to prevent any pathological effect in the event of re-exposure or first exposure. Detailed description of the invention

[0022] Antibody according to the invention

[0023] To identify an anti-ricin antibody more effective than those already existing, the inventors immunized BALB / c mice with inactivated ricin (isoform E), then, once the immune response was triggered, with non-inactivated ricin (isoform E). The splenic B lymphocytes of these mice were collected and fused with NSI cells to form hybridomas, secreting antibodies in the culture supernatant. They then selected several hybridomas by screening their supernatants using EIA (Enzyme Immuno-Assays) tests in order to evaluate the specificity of the antibodies secreted by these hybridomas with respect to the ricin D and E isoforms of the cultivar R. carmencita (the ultimate objective being to obtain antibodies capable of recognizing both isoforms indifferently). The hybridomas whose supernatants gave the best responses in EIA were then cloned in limiting dilution, in order to obtain monoclonal populations of hybridomas. The neutralizing capacity of the antibodies present in these supernatants was subsequently tested with Jurkat cells placed in the presence of dilutions of these supernatants and a cytotoxic dose of ricin (approximately 10 CD50: 10 times the dose of ricin capable of causing 50% cell death). The hybridoma secreting the antibody named ERA5 was finally selected because it had an excellent capacity for specific neutralization of both isoforms D and E of ricin.

[0024] As demonstrated by the examples below, ERA5 has equivalent affinities for both D and E isoforms, and of the same order of magnitude as the most affinity antibodies published, i.e. a KD of the order of picomolar (measured by BLI). In addition, its neutralization and protection performances are much superior to those of already existing antibodies. Although the results are difficult to compare, on average, after four identical repetitions of the experiment, 92% survival was observed in mice exposed to 5LD50 of ricin intranasally (~2 pg per mouse) and treated 6 hours later with the ERA5 antibody at 10 mg / kg iv. Three of these experiments demonstrated a survival rate of 100%.These results were obtained by measuring the survival of mice for 21 days, a longer duration than that analyzed in the publications (see Table 2), and less favorable in terms of survival (it often happens that experiments are stopped after 6 to 14 days in the publications, even though animals still die beyond 14 days - which favors the survival data presented). Survival rates of 50% and 35% were also observed with ERA5 (10 mg / kg) injected iv 18h and 24h respectively after exposure to 5LD50 of ricin. These survival rates are higher than all those published so far with monoclonal antibodies and for such a wide therapeutic window.

[0025] The surviving mice from these ERA5-induced protection experiments were re-exposed to the same dose of ricin (5 LD50) 10 to 12 months after the first intoxication, without any associated treatment. The survival rate of the mice in these late re-exposure experiments was 100%, regardless of the time interval between ricin exposure and the administration of the antibody(ies) during the first experiment ([Fig.7]). In addition, a less severe clinical outcome at the second exposure could be observed. In summary, the mice appear physiologically less affected by the second intoxication, and long-term active immunity to ricin screw was therefore induced after treatment of the poisoning using our antibodies.

[0026] The present invention aims to protect the ERA5 antibody identified by these experiments, as well as its functional derivatives and all prophylactic and therapeutic uses involving it.

[0027] As is well known, only a part of the antibody, the variable region, is involved in the binding of the antibody to its epitope. The constant regions of the antibody activate immune effectors, including phagocytes, killer cells and complement, as well as other receptors such as the Brambell receptor involved in the biological recycling of antibodies; these constant regions are not involved in antigen binding. Within the variable region are the complementary determining regions (CDRs), also called hypervariable regions, which interact directly with the antigen. Modifying the CDRs can therefore modify the affinity of an antibody. The CDR3 regions belonging to the variable regions of the heavy chain and the light chain are particularly important for antigen recognition.

[0028] The CDRs of the antibody of the invention are summarized in Table 3 below:

[0029] [Tables 3] SEQ ID NO Amino acid sequence CDR1 VL 1 QDISNY CDR2 VL 2 YTS CDR3 VL 3 QQYSKRLPT CDR1 VH 4 GYSITSDYY CDR2 VH 5 ISYDGSN CDR3 VH 6 ARDAHYSNYFDF

[0030] Table 3: CDR of the antibody according to the invention

[0031] The sequence SEQ ID NO: 2 does not have the minimum length required according to the WIPO ST26 standard. It is therefore entered as an omitted sequence in the XML sequence listing. Consequently, the sequence itself is not entered in the sequence list; instead of the sequence, the indication "000" appears. The sequence SEQ ID NO: 2 is nevertheless part of the invention and has the sequence YTS, as indicated in Table 3 above.

[0032] According to a first aspect, the present invention relates to an antibody or a functional fragment of antibody, isolated and purified, capable of recognizing and neutralizing the ricin isoforms D and E, and comprising, or consisting essentially of, or consisting of: a. a light chain comprising the CDRs having the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or having a sequence identity of at least 80% with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and b. a heavy chain comprising the CDRs having the sequences SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 or having a sequence identity of at least 80% with SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0033] The functional fragments of a given antibody are well known to those skilled in the art. An antibody whose constant region (Fc) has been enzymatically cleaved so as to preserve the hinge region is referred to as an F(ab')2 fragment and retains both antigen-binding sites. Similarly, an antibody whose constant region, including the hinge region, has been enzymatically cleaved, or which has been produced without this region, is referred to as a Fab fragment and retains one of the two antigen-binding sites. Fab fragments consist of a light chain that is covalently linked to a portion of the heavy chain called Fd. An scFv fragment consists of the variable region of the heavy chain and the variable region of the light chain linked together by a peptide bond that allows the two variable regions to associate to form an antigen-binding site.

[0034] In certain embodiments of the invention, Fab or scFv fragments are preferred for the following reasons: a) because Fab or scFv fragments have only one antigen binding site, large immune complexes cannot form, b) the absence of an Fc region prevents the occurrence of an Fc-activated inflammatory reaction, such as activation of the complement cascade, c) tissue penetration of a small Fab or scFv molecule is easier, and d) Fab or scFv production is easily and inexpensively performed in bacteria such as E. coli.

[0035] Thus, an object of the present invention is to provide Fabs of the anti-ricin antibody according to the invention, fragments of this antibody smaller or larger than Fab fragments or epitope-binding peptides, and in particular peptides derived from the hypervariable regions of the anti-ricin antibody according to the invention.

[0036] The light chain of the murine ERA5 antibody has the sequence (highlighted CDRs):

[0037] DIOMTOTTSSLSASLGDRVTISCRASODISNYLNWYQQKPDGTVKLLIYYTS RLHSGVPSRFSGSGSGTEYSLTISNLEPEDIATYYCOQYSKRLPTFGGGTKLEIK = SEQ ID NO: 7

[0038] The heavy chain of the murine ERA5 antibody has the sequence (highlighted CDRs):

[0039] DVOLQESGPGLVKPSOSLSLTCSVTGYSITSDYYWNWIROFPGNKLEWMGY ISYDGSNNYNPSLNNRISITRDVIKNQFFLKLNSVTTEDTATYYC ARDAHYSNYFDFWGOGTTLTVSS = SEQ ID NO: 8

[0040] Preferably, the antibody or antibody fragment of the invention is characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 7, or an amino acid sequence having at least 80% identity with SEQ ID NO: 7.

[0041] Preferably, the antibody or antibody fragment of the invention is characterized in that its heavy chain contains the variable domain VH of sequence SEQ ID NO: 8, or an amino acid sequence having at least 80% identity with SEQ ID NO: 8.

[0042] The inventors were able to demonstrate that the affinity of the ERA5 antibody towards ricin D and towards ricin E of the cultivar R. carmencita is less than 10 12, when measured for example by bi-layer interferometry (BLI).

[0043] Thus, the antibody or antibody fragment of the invention is preferably characterized in that its affinity with ricin D and / or with ricin E of the cultivar R. carmencita is between 109 and 10 12, or even less than 10 12, measured by bi-layer interferometry (BLI).

[0044] The present invention also provides chimeric antibodies in which the Fc portion and / or constant regions of the antibody are derived from human or non-human homologous sequences. According to one embodiment of the invention, the Fc portion of the antibody may be selected so as to produce IgA, IgM or IgG.

[0045] According to another embodiment of the invention, the Fc portion of the antibody may be an Fc portion originating from mice, horses, sheep, cattle or other mammals. The present invention also covers scFv fragments fused to Fc regions.

[0046] After discovering the excellent protection induced by the ERA5 antibody against ricin poisoning, the inventors undertook to humanize the polypeptide sequences of the murine ERA5 antibodies with a view to being able to administer them to humans.

[0047] Also included by antibodies according to the present invention are “chimeric” or “humanized” antibodies. By “chimeric” antibody is meant an antibody which contains a natural variable region (light chain and heavy chain) derived from an antibody of a given species in association with the constant regions of light chain and heavy chain of an antibody of a species heterologous to said given species. The antibodies or their fragments of chimeric type according to the invention can be prepared using genetic recombination techniques. For example, the chimeric antibody can be produced by cloning a recombinant DNA comprising a promoter and a sequence coding for the variable region of a non-human, in particular murine, monoclonal antibody according to the invention, and a sequence coding for the constant region of a human antibody. A chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from murine DNA and its isotype determined by the constant region derived from human DNA. For methods of preparing chimeric antibodies, reference may be made, for example, to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

[0048] By "humanized" antibodies is meant an antibody that contains CDR regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies. In addition, some of the residues of the backbone segments (referred to as FRs) may be modified to retain binding affinity (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988). The humanized antibodies according to the invention or their fragments can be prepared by techniques known to those skilled in the art (such as, for example, those described in the documents Singer et al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; or Bebbington et al., Bio / Technology, 10:169-175, 1992).Such humanized antibodies according to the invention are preferred for their use in prophylactic and / or therapeutic treatment in vivo. Other humanization techniques are also known to those skilled in the art, such as, for example, the “CDR Grafting” technique described by PDL, which is the subject of patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761.

[0049] According to a preferred embodiment of the invention, the anti-ricin antibody according to the invention has an Fc portion of human origin. Such whole antibodies are preferred for administration in humans because they have a longer half-life than antibody fragments such as Fabs, and are more suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal or aerosol administration.

[0050] As shown in the examples below, a murine / human chimeric form was recombinantly produced in ExpiCHO cells. This version of ERA5 has murine variable parts and constant CH1, CH2, CH3 fragments of human antibodies. The neutralizing capacity of the initial ERA5 antibody was not impacted by this modification ([Fig.8]).

[0051] A first batch of humanized antibody from ERA5 was then produced (version 1 = vl). The light and heavy chains of the humanized antibody vl produced by the inventors have the following sequences (mutations relative to the murine ERA5 antibody sequences are underlined, while CDRs are highlighted in gray):

[0052] Light chain (LC):

[0053] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAP KLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKRLPTFG QGTKVEIK = SEQ ID NO: 9

[0054] Heavy chain (HC):

[0055] QVQLQESGPGLVKPSQTLSLTCSVSGYSITSDYYWNWIRQPPGKG LEWIGYISYDGSNNYNPSLNNRISISVDTSKNOFSLKLNSVTAADTAV YYCARDAHYSNYFDFWGQGTLVTVSS = SEQ ID NO: 10

[0056] This first humanized form vl, produced recombinantly, neutralizes ricin in vitro but with an IC50 10 times higher than its murine parent ([Fig.8]). In order to compensate for this loss of neutralization, affinity maturation by molecular engineering was carried out on the humanized version of ERA5.

[0057] A new humanized version was therefore produced: it is the antibody “humanized ERA5 v2” whose chain sequence takes up the sequence of humanized ERA5 v1, but with 2 substitutions in the variable heavy chain: S71T and V72R.

[0058] A second batch of humanized antibody from ERA5 was then produced (version 2 = v2). The light and heavy chains of the humanized antibody v2 produced by the inventors have the following sequences (mutations compared to the sequences of the murine ERA5 antibody are underlined, while the CDRs are highlighted in gray):

[0059] Light chain (LC, identical to that of vl):

[0060] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAP KLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKRLPTFG QGTKVEI K = SEQ ID NO: 9

[0061] Heavy chain (HC, different from vl by S71T and V72R)

[0062] QVQLQESGPGLVKPSQTLSLTCSVSGYSITSDYYWNWIRQPPGKG LEWIGYISYDGSNNYNPSLNNRISITRDTSKNQFSLKLNSVTAADTAy YYCARDAHYSNYFDFWGQGTLVTVSS = SEQ ID NO: 11

[0063] Interestingly, when 1TC5o of the humanized antibody v2 is compared with 1TC5o of the murine antibody ERA5 and that of vl, it is found that the neutralizing capacity of the humanized antibody v2 is better than that of the humanized antibody vl (although less than that of the murine antibody ERA5 described above).

[0064] The ERA5 antibody is a murine monoclonal antibody. To be administrable in humans, the antibody of the invention is preferably a chimeric, humanized or human antibody obtained from the human ERA5 antibody by conventional antibody humanization techniques. The antibody of the invention may also be a polyclonal antibody, provided that its CDRs have the sequences SEQ ID NO: 1-6, or are at least 80% identical to SEQ ID NO: 1-6.

[0065] The present invention preferably relates to an anti-ricin antibody according to the invention which has been humanized, in the sense that it comprises, in the variable region of the heavy chain, in comparison with the sequence SEQ ID NO: 8 of murine ERA5, at least one, preferably two, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or even 17 mutations selected from the group consisting of:

[0066] -D / Q (1): amino acid Q (glutamine) replaces amino acid D (aspartic acid) at position 1

[0067] - S / T (17): the amino acid T (threonine) replaces the amino acid S (Serine) at the position 17.

[0068] - T / S (25): the amino acid S (serine) replaces the amino acid T (threonine) at position 25.

[0069] - F / P (41): the amino acid P (proline) replaces the amino acid F (Phenylalanine) at the position 41.

[0070] - N / K (44): the amino acid K (lysine) replaces the amino acid N (Asparagine) at the position 44.

[0071] - K / G (45): amino acid G (glycine) replaces amino acid K (lysine) at position 45.

[0072] - M / I (49): amino acid I (isoleucine) replaces amino acid M (methionine) at the position 49.

[0073] - T / S (71): the amino acid S (serine) replaces the amino acid T (threonine) at position 71.

[0074] - R / V (72): amino acid V (valine) replaces amino acid R (arginine) at position 72.

[0075] - V / T (74): amino acid T (threonine) replaces amino acid V (valine) at the position 74.

[0076] - I / S (75): amino acid S (serine) replaces amino acid I (isoleucine) at position 75.

[0077] - F / S (80): the amino acid S (serine) replaces the amino acid F (phenylalanine) at the position 80.

[0078] - T / A (88): amino acid A (alanine) replaces amino acid T (threonine) at the position 88.

[0079] - E / A (89): : amino acid A (alanine) replaces amino acid E (glutamic acid) at position 89.

[0080] - T / V (93): amino acid V (valine) replaces amino acid T (threonine) at the position 93.

[0081] - T / L (115): the amino acid L (leucine) replaces the amino acid T (threonine) at the position 115.

[0082] - L / V (116): amino acid V (Valine) replaces amino acid L (leucine) at the position 116.

[0083] In another embodiment of the invention, the anti-ricin antibody according to the invention is humanized, in the sense that it comprises, in the variable region of its light chain, in comparison with the sequence SEQ ID NO: 7 of murine ERA5, at least one, preferably two, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations selected from the group consisting of:

[0084] - T / S (7): the amino acid S (serine) replaces the amino acid T (threonine) at position 7.

[0085] - T / P (8): the amino acid P (proline) replaces the amino acid T (Threonine) at the position 8.

[0086] - L / V (15): amino acid V (valine) replaces amino acid L (leucine) at position 15.

[0087] - S / T (22): the amino acid T (threonine) replaces the amino acid S (Serine) at the position 22.

[0088] - D / G (41): amino acid G (Glycine) replaces amino acid D (aspartic acid) at position 41.

[0089] - G / K (42): amino acid K (lysine) replaces amino acid G (glycine) at position 42.

[0090] - T / A (43): amino acid A (alanine) replaces amino acid T (threonine) at the position 43.

[0091] - V / P (44): amino acid P (proline) replaces amino acid V (valine) at position 44.

[0092] - E / D (70): amino acid D (aspartic acid) replaces amino acid E (aspartic acid) glutamic acid) at position 70.

[0093] - S / T (72): amino acid S (serine) replaces amino acid E (glutamic acid) at the position 72.

[0094] - N / S (77): the amino acid N (asparagine) replaces the amino acid S (Serine) at the position 77.

[0095] - E / Q (79): amino acid E (Glutamic acid) replaces amino acid Q (glutamine) at position 79.

[0096] - I / F (83): amino acid I (isoleucine) replaces amino acid F (phenylalanine) at the position 83.

[0097] - G / Q (100): amino acid Q (glutamine) replaces amino acid G (glycine) at the position 100.

[0098] - L / V (104): amino acid V (valine) replaces amino acid L (leucine) at position 104.

[0099] In a preferred embodiment of the invention, the anti-ricin antibody according to the invention, when humanized, comprises at least one, preferably two, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or even 17 mutations as defined above in the variable region of its heavy chain as described above and at least one, preferably two, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or even 15 mutations as defined above in the variable region of its light chain as described above.

[0100] In a particular embodiment, the antibody or antibody fragment according to the invention is humanized or completely human, and is characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 9, or an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and in that its heavy chain contains the variable domain VH of sequence SEQ ID NO: 10, or an amino acid sequence having at least 80% identity with SEQ ID NO: 10.

[0101] In a particular embodiment, the antibody or antibody fragment according to the invention is humanized or completely human, and is characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 9, or an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and in that its heavy chain contains the variable domain VH of sequence SEQ ID NO: 11, or an amino acid sequence having at least 80% identity with SEQ ID NO: 11.

[0102] Variants of the amino acid sequence of the antibody of the present invention may be prepared by using only a portion of the nucleotide or peptide sequences presented in the present text. Such variants comprise, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. The present invention relates to any variant obtained from the sequences presented in this text provided that the expression of the final construct has the expected characteristics of an anti-ricin antibody according to the invention, namely an antibody capable of binding to the E and D isoforms of ricin of several cultivars with an affinity of at most 10 9M.In general, the amino acid sequence of the antibody variants of the present invention has at least 80% sequence identity with the original anti-ricin antibody variable region sequence, preferably at least 85%, 87%, 90% and more preferably 95% sequence identity with the original anti-ricin antibody ERA5 variable region sequence described above.

[0103] Examples of variants according to the invention are, for example, variants resulting from the insertion of amino acids in the N or C-terminal position, ranging from one residue to Polypeptides comprising more than 100 residues, as well as the insertion within the sequence of one or more amino acids. Examples of terminal insertions are an antibody with an N-terminal methionine residue or an antibody fused with an epitope tag (HA, c-myc, polyhistidine sequence, HSV glycoprotein D) or an antibody fused to a polypeptide that increases the neutralizing capacity of the antibody.

[0104] Examples of variants according to the invention are, for example, variants resulting from the substitution of certain amino acids originally present in the antibodies of the invention, by amino acids more favorable to their production or to their biological effect. These variants are for example obtained by substitutional mutagenesis in the hypervariable or framework regions of the antibody. Substantial modifications in the biological properties of the antibody of the present invention can be obtained by selection of substitutions which differ significantly in their effect on the maintenance of (a) the structure of the polypeptide backbone at the level of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain.

[0105] Natural residues can be divided into different groups according to their properties:

[0106] (1) hydrophobic residues: norleucine, met, ala, val, leu, ile;

[0107] (2) hydrophilic neutral residues: cys, ser, thr;

[0108] (3) acid residues: asp, glu;

[0109] (4) basic residues: asn, gin, his, lys, arg;

[0110] (5) residues influencing chain orientation: gly, pro; and

[0111] (6) aromatic residues: trp, tyr, phe.

[0112] Conservative substitutions are well known. They involve the replacement of an amino acid with another member of the same group (said group being one of groups (1) to (6) as defined above), while non-conservative substitutions involve the replacement of one of the groups with a member of another group.

[0113] Examples of conservative substitutions within these six groups, but also other examples of conservative substitutions, are shown in Table 4 below.

[0114] [T ables 4] Original amino acid Possible substitutions Preferred substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gin, Asn Lys Asn (N) Gin, His, Asp, Lys, Gin Asp Asp (D) Glu, Asn Glu Cys (C) Ser, Ala Ser Gin (Q) Asn, Ala Ser Glu (E) Asp, Gin Asp Gly (G) Ala Ala His (H) Asn, Gin, Lys, Arg Ile (D Leu, Val, Met, Ala, Phe Leu, Norleucine Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gin, Asn Arg Met (M) Leu, Phe, Ile Leu Phe(F) Leu, Val, Ile, Ala, Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr(Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norle ucine Leu

[0115] Table 4: Examples of conservative substitutions

[0116] Any cysteine ​​residue not involved in maintaining the conformation of the antibody can also be substituted, generally with a serine, to increase the oxidative stability of the molecule. Similarly, cysteine ​​bonds can be added to increase the stability of the antibody (particularly in the case of Fv fragment).

[0117] The preferred variants according to the invention are, for example, variants resulting from one or more mutations inducing a conservative substitution.

[0118] The variants according to the invention may also have been obtained by affinity maturation. Affinity maturation involves the preparation and screening of antibody variants that possess mutations (deletions, insertions, or substitutions) in the variable regions of the parental antibody, as well as the selection of variants having improved biological properties such as better binding affinity compared to the parental antibody or better tolerance during administration. to humans. A conventional method for generating such affinity-enhanced variants is affinity maturation using mutagenesis techniques followed by selection, for example by phage display.

[0119] Immunoconjugates

[0120] The present invention also relates to an immunoconjugate comprising an anti-ricin antibody according to the invention linked, directly or indirectly, to a therapeutic agent.

[0121] Such therapeutic agents include chemical agents, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, or enzyme inhibitors. Examples of toxins are diphtheria A chain, exotoxin A chain, Tabrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes. Examples of radionuclides are Bi, I, In, Y, and 186Re.

[0122] The inventors have identified an immunoconjugate with particularly advantageous properties. This is a bispecific “ERA5-E5” antibody, which is a bispecific version where VHH E5 (public sequence of VHH E5, as described in Rudolph et al., 2014 (

[11] , https: / / doi.Org / 10.1016 / j.jmb.2014.05.026)), and whose structure is described in the Protein Data Bank (PDB) database under number 4LGP (https: / / www.rcsb.org / structure / 4LGP))) has been grafted into the C-terminal position of the heavy chain of humanized ERA5 v2 described above. They demonstrated that the neutralizing capacity of the bispecific humanized antibody v2 coupled with E5 is much better than that of the initial murine ERA5 antibody, and, a fortiori, that of the monospecific humanized antibodies v1 and v2. Grafting VHH E5 onto humanized ERA5 v2 therefore significantly improves its neutralizing capacities, even exceeding those of the initial murine version.

[0123] The VHH E5 antibody has the sequence (PDB 4LGP):

[0124] QVQLVETGGGLVQPGGSLTLSCAGSGGTLEHYAIGWFRQAPGKEHEWLVCN RGEYGSTVYVDSVKGRFTASRDNAKNTVYLQLNSLKPDDTGIYYCVSGCYS WRGPWGQGTQVTVSS = SEQ ID NO: 12

[0125] In a particular embodiment, the present invention therefore relates to an antibody or antibody fragment according to the invention, said antibody being bispecific and coupled to the VHH E5 antibody;

[0126] preferably wherein said VHH E5 antibody comprises, or consists essentially of, or consists of, a sequence preferably having at least 80% identity with the sequence SEQ ID NO: 12, more preferably at least 85% identity”, more preferably at least 90% identity, more preferably at least 95% identity with the sequence SEQ ID NO: 12;

[0127] more preferably, said VHH E5 antibody comprises, or consists essentially of, or consists of, the sequence SEQ ID NO: 12.

[0128] Hybridoma

[0129] In another aspect, the present invention protects the hybridoma which makes it possible to produce the murine ERA5 antibodies of the invention, as described above. This hybridoma was deposited in the National Collection of Cultures of Microorganisms of the Pasteur Institute, (having the address CNCM, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15), on February 14, 2024, under the number CNCM 1-6035.

[0130] It produces the murine ERA5 monoclonal antibody of the invention.

[0131] The present invention also relates to the monoclonal antibody produced by this hybridoma.

[0132] Nucleic acids and host cells

[0133] According to the above description of the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain of the anti-ricin antibodies according to the invention, a person skilled in the art is capable of synthesizing, or having synthesized, nucleic acids which encode these amino acid sequences.

[0134] The subject of the present invention is therefore a nucleic acid encoding an anti-ricin antibody according to the invention, or for one of its functional or immunoconjugated fragments.

[0135] More particularly, the present invention therefore relates to an isolated nucleic acid, chosen from:

[0136] a) a nucleic acid, DNA or RNA, coding for the antibody or one of its fragments or immunoconjugates as defined in the present description;

[0137] b) a nucleic acid present in the cells deposited in the National Collection of Microorganism Cultures of the Pasteur Institute, on February 14, 2024, under the number CNCM 1-6035, said nucleic acid coding for the antibody or one of its fragments as defined in the present description; and

[0138] c) a nucleic acid whose sequence has a percentage identity of at least 80% after optimal alignment with one of the sequences a) or b).

[0139] These nucleic acids may be included in a recombinant vector for cloning or for expressing the antibodies of the invention.

[0140] The present invention also relates to a vector comprising said nucleic acid. These nucleic acids may be included in a recombinant vector for cloning or for expressing the antibodies of the invention. These vectors are usually introduced into host cells to enable their cloning or expression.

[0141] The present invention therefore also relates to a vector or a host cell comprising one of the nucleic acids described above.

[0142] The present invention includes all recombinant vectors containing coding sequences for eukaryotic or prokaryotic transformation, transfection or gene therapy. Such vectors may be prepared according to conventional molecular biology techniques and will further comprise a suitable promoter, optionally a signal sequence for export or secretion, and regulatory sequences necessary for transcription of the nucleotide sequence. A fusion polypeptide may be useful for the purification of the antibodies of the present invention. The fusion domain may for example include a poly-histidine tail which allows purification on Ni+ columns, or a filamentous phage membrane anchor which is particularly useful for library screening, according to "phage display" technology.

[0143] The term "vector" refers to a nucleic acid into which the sequence of interest can be inserted by restriction and then ligation for transport between different genetic environments or for expression in a host cell. Vectors are, for example, plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and bacteriophage P 1-derived artificial chromosomes (PACs), virus-derived vectors. A cloning vector is a vector capable of replicating in a host cell and which is further characterized by the presence of one or more endonuclease restriction sites. An expression vector is a vector into which the DNA sequence of interest can be inserted by restriction or ligation in such a way that it can be replicated and / or transcribed into RNA.The vectors may further contain one or more markers for selection or identification of cells that have been transformed or transfected with the vector.

[0144] One of the vectors suitable within the scope of the invention is a recombinant DNA molecule adapted to receive and express a first and a second DNA sequence, so as to allow the expression of heterodimeric antibodies such as a full-length antibody or F(ab')2 or Fab fragments according to the invention. Such a vector provides a system for independently cloning the two DNA sequences into two separate cassettes present in the vector, so as to form two separate cistrons for the expression of a first and a second polypeptide of the heterodimeric antibody. Such an expression vector is called a di-cistronic vector.

[0145] The modified antibodies of the present invention can be produced in eukaryotic cells such as CHO or plant cells.

[0146] Compositions comprising the antibody according to the invention

[0147] Another object of the present invention is to provide a composition comprising at least one anti-ricin antibody according to the invention, or one of its functional fragments, or immunoconjugates, as described above.

[0148] Preferably, this composition is a pharmaceutical composition, which contains, in addition to the antibody or antibody fragment or immunoconjugate of the invention, a pharmaceutically acceptable excipient.

[0149] In other words, the present invention therefore relates to a medicament comprising the antibody or the antibody fragment or the immunoconjugate of the invention and a pharmaceutically acceptable excipient.

[0150] In the present description, the term "pharmaceutically acceptable excipient" is intended to denote a compound or a combination of non-toxic compounds included in a pharmaceutical composition which 1) does not interfere with the effectiveness of the biological activity of the active ingredients of the composition and 2) does not cause side reactions and which allows, for example, the facilitation of the administration of the antibody of the invention, the increase of its lifespan and / or its effectiveness in the body, the increase of its solubility in solution or even the improvement of its preservation. They are compatible with biological systems such as a cell, a cell culture, a tissue or an organism. These pharmaceutically acceptable excipients are well known and will be adapted by those skilled in the art according to the nature and the method of administration of the active compound(s) chosen.

[0151] Prevention / treatment

[0152] The present invention also relates to one of these compositions for its use to protect against (in the case of preventive administration of the antibody) or to reduce (in the case of treatment administered after intoxication) the effects of ricin intoxication.

[0153] Furthermore, it relates to the use of an antibody according to the invention, or of one of its functional fragments, or one of its immunoconjugates, to prepare a medicament intended for the treatment or prevention of ricin poisoning.

[0154] The present invention also relates to a method for treating a subject, preferably a human, likely to be poisoned by ricin, in which a therapeutically effective amount of an antibody according to the invention, or of one of its functional fragments, or of one of its immunoconjugates is administered to said subject. A therapeutically effective amount corresponds to an amount sufficient to reduce the symptoms and the progression of the poisoning. This amount may vary with the age, the sex of the subject and the stage of the poisoning and will be determined by a person skilled in the art. A therapeutically effective amount of the antibody according to the invention, or of one of its functional fragments, or of one of its immunoconjugates, may vary between 0.01 mg / kg and 50 mg / kg. preferably between 0.1 mg / kg and 20 mg / kg, and more preferably between 0.1 mg / kg and 10 mg / kg, in one or more administrations, for one or more days.

[0155] The term "ricin poisoning" refers to any disease caused, directly or indirectly, by the intentional or accidental ingestion, infusion, inhalation, or cutaneous application of ricin by an individual. Ricin does not show any selectivity for a specific cell type. Since all cell types can be affected, the symptoms depend on the site of entry of the toxin into the human body. Depending on the dose, the first symptoms appear after a latency period of 2 to 24 hours. After inhalation of a large dose of ricin, the victim may experience general disorders (fever, sweating, nausea) and breathing difficulties, including pulmonary edema. Disorders of gas exchange can lead to death. After oral ingestion of ricin, the victim suffers from vomiting and diarrhea that can become bloody, causing severe dehydration or even collapse.Within a few days, splenic, hepatic, and renal functions can be significantly impaired. Death from ricin poisoning can occur within 36 to 72 hours after poisoning, depending on the dose of ricin and the route of poisoning.

[0156] The method of administration may be by injection or by gradual infusion. The injection may be intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal. Another method of administration may be by aerosol.

[0157] Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol / water solutions, emulsions or suspensions.

[0158] As used herein, the term "treat" or "treatment" means an improvement in the patient's disease, disorder, or condition, which can be observed clinically, histologically, and / or biochemically after ricin has been ingested or administered to the patient. The terms "treat" or "treatment" include, but are not limited to, the non-development or improvement of a symptom or clinical, histological, and / or biochemical parameter associated with the patient's disease, disorder, or condition or the inhibition, reduction, or delay of the progression or exacerbation of the patient's disease, disorder, or condition (including secondary damage caused by the disease, disorder, or condition) either to a statistically significant degree or to a degree detectable by those skilled in the art, once ricin has been administered to the patient.

[0159] As used herein, the terms "prevent", "prevention" and "prevent" refer to reducing the risk of a subject acquiring or developing symptoms of ricin poisoning when brought into contact with ricin. The terms "prevent", "prevention" and "prevent" also include delaying the onset and / or reducing the frequency and / or intensity of clinical, histological and / or biochemical symptoms or parameters associated with subsequent ricin poisoning.

[0160] In particular, the compositions and methods of the invention make it possible to delay or even prevent the symptoms of a second ricin poisoning after a first poisoning ([Fig.7]). In this case, the principle of prevention applies to the second poisoning, although the administration of the compositions of the invention also makes it possible to “treat” the first poisoning. Thus, the compositions and methods of the invention, when used between two contacts with ricin, are both prophylactic and therapeutic.

[0161] Uses and kits for detecting ricin

[0162] According to a particular embodiment, the anti-ricin antibody of the invention can be used in vitro, for example in immunological diagnostic tests in which they are used in liquid phase or bound to a solid phase vehicle. Examples of well-known vehicles are glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamide, agarose or magnetite. Examples of immunological tests using the antibody of the invention are radioimmunoassays, histoimmunological stainings, EL1SA, western blots, immunoprecipitation tests, immunodiffusion tests, complement fixation tests, FACS analyses or protein microarray analyses.

[0163] The present invention also aims to provide methods for the in vitro detection of ricin in a biological sample, said method comprising: bringing the sample into contact with at least one anti-ricin antibody according to the invention, and detecting said anti-ricin antibody as an indicator of the presence of said ricin. The biological sample may be liquid: for example saliva, urine, cerebrospinal fluid, serum or blood, or solid or semi-solid, for example tissues or feces or a solid tissue as commonly used in histological diagnosis.

[0164] In this type of applications, the anti-ricin antibody according to the invention can be labeled. Examples of labels include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Methods for binding a label to an antibody are well known to those skilled in the art. Another labeling technique consists of coupling the antibody to low molecular weight haptens, which can be specifically modified by means of a second reaction. Examples of haptens are biotin, which reacts with avidin, or dinitrophenol, pyridoxal or fluorescein, which can react with specific anti-hapten antibodies.

[0165] The present invention also aims to provide a method for in vivo detection of ricin, in which a labeled anti-ricin antibody according to the present invention is administered to a subject. The amount of labeled antibody administered must be sufficient to allow detection of the binding of the antibody to the toxin. The amount of labeled antibody administered will depend on factors such as the age and sex of the subject, as well as the stage of the disease. The amount administered may vary between 0.01 mg / kg and 50 mg / kg, preferably between 0.1 mg / kg and 20 mg / kg, and more preferably between 0.1 mg / kg and 2 mg / kg.

[0166] To perform in vivo diagnosis, the anti-ricin antibody of the invention may be linked to a radioisotope directly or indirectly via functional groups. Commonly used functional groups are, for example, diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA). Examples of radioisotopic metal ions are 1HIn, 97Ru, 67Ga,68 "70 Q Û OH 1 Ga, As, Zr and Tl. The anti-ricin antibodies of the invention can also be labeled with a paramagnetic isotope for diagnosis by magnetic resonance imaging (MRI) or electron spin resonance (ESR). Radioisotopes 1 <"7 cc 1 AO Positron emitters can also be used, such as Gd, Mn, Dy, 68Ga, 52Cr, and 56Fe.

[0167] The anti-ricin antibodies of the invention can also be used in vitro or in vivo to monitor the progress of disease treatment, for example by determining the increase or decrease in the number of cells targeted by ricin or changes in the concentration of ricin in a biological sample.

[0168] An object of the present invention is finally to provide a kit for the in vitro or in vivo detection of ricin. This kit may contain in particular: a container comprising at least one anti-ricin antibody according to the invention, which may be (or not) labeled, optionally, a container comprising buffer solutions and optionally a container comprising means for detecting said labeled anti-ricin antibody, such as a biotin-binding protein (for example avidin or streptavidin) optionally linked to a detectable marker, such as a fluorescent or enzymatic marker. This container may also comprise means for detecting said unlabeled anti-ricin antibody, in practice for example antibodies or antibody fragments. Figure legends

[0169] [Fig. 1]: Neutralization curves of equimolar ricin D+E by ERA5 compared to an antibody described in the literature: RB34. For the latter, each point corresponds to the average over the technical triplicate of the experiment, the error bars are the standard deviation over these triplicates. For ERA5, each point corresponds to the average over more than three separate experiments and the error bars are the corresponding standard deviations. The antibodies were administered with 10CD50 of ricin D+E on Jurkat cells. Viability was measured 72 hours later.

[0170] Figure 2: Graphs representing the kinetic parameters of the murine antibody ERA5 measured by BLI. (Figure 2 A) Ricin isoform D binding curve; (Figure 2 B) Ricin isoform E binding curve.

[0171] [Fig.3]: Ricin D / E neutralization curves (equimolar proportions) by ERA5 on different cell types. Each point on the curves corresponds to the average of at least three separate experiments. Error bars correspond to the standard deviation.

[0172] [Fig.4]: Neutralization of ricin from different preparations and cultivars by ERA5. The equimolar mixture of ricins D and E purified from the cultivar R. carmencita is used as a reference for comparison. The neutralization of ricin from the different cultivars by ERA5 is not significantly different from the neutralization of the reference ricin (non-parametric Kruskal-Wallis test).

[0173] [Fig.5]: Percentage of survival of mice treated with ERA5 (10 mg / kg) at different treatment time (6h, 10h, 18h and 24h) after intoxication with 5 LD50 of ricin D + E (100 μg / kg) These curves represent the average of the survival percentages carried out on several separate experiments, the number of which is not specified in the graph legend. Each condition was tested on at least 10 BALB / c mice. The ricin control group corresponds to the groups that received 5 LD50 of ricin in followed by no treatment. This control was carried out with 5 or 10 mice for each separate experiment. The Mantel-Cox statistical test was applied to the treated groups compared to the ricin control group: * p < 0.05; ** p < 0.01 *** p < 0.001; **** p < 0.0001; ns: not significant.

[0174] [Fig.6]: Percentage of survival of mice treated with different antibody cocktails (10 mg / kg iv) 6 h after intoxication with 5LD50 of ricin D+E (100 µg / kg in). These survival percentages were calculated from groups of 10 BALB / c mice in one or more separate experiments (number of repetitions n specified in the legend). The Mantel-Cox statistical test was applied to the treated groups compared to the ricin control group: * p < 0.05; ** p < 0.01 *** p < 0.001; **** p < 0.0001; ns: not significant.

[0175] [Fig.7]: Percentage of survival of mice re-intoxicated, for the second time, with 5LD50 of ricin, 10 months after their first exposure. In the legend of the graph is indicated the treatment that 10 BALB / c had received during their first intoxication (10 mg / kg of iv antibodies). For each group, the number of re-intoxicated mice was: ERA5 6h: 7; ERA5 10h: 3, ERA5 18h: 5; ERA5 24h: 4. The Mantel-Cox statistical test was applied to the treated groups compared to the ricin control group: * p < 0.05; ** p < 0.01 *** p < 0.001; **** p < 0.0001; ns: not significant.

[0176] [Fig.8]: Neutralization curves of ricin D+E by different forms of ERA5 (humanized = vl). Each point corresponds to the average of 3 technical replicates and the error bars are the associated standard deviations.

[0177] [Fig.9]: Neutralization curves of ricin by different forms of ERA5. Examples

[0178] Production of the antibody of the invention

[0179] ERA5 is an antibody derived from BALB / c mice first immunized with inactivated ricin (isoform E), then once the immune response was triggered, with non-inactivated ricin isoform E. Splenic B lymphocytes from these mice were collected and fused with NSI cells to form hybridomas, secreting antibodies into the culture supernatant.

[0180] The hybridomas were then selected by screening their supernatants using EIA (Enzyme Immuno-Assays) tests in order to evaluate the specificity of the antibodies secreted with respect to the ricin D and E isoforms of the cultivar R. carmencita (the ultimate objective being to obtain antibodies capable of recognizing both isoforms indifferently). The hybridomas whose supernatants gave the best responses in EIA were then cloned in limiting dilution, in order to obtain monoclonal populations of hybridomas. The neutralizing capacity of the antibodies present in these supernatants was subsequently tested with Jurkat cells placed in the presence of dilutions of these supernatants and a cytotoxic dose of ricin (approximately 10 CD50: 10 times the dose of ricin capable of causing 50% cell death). The ERA5-secreting hybridoma was finally selected because it exhibited excellent specific neutralization capacity for ricin.

[0181] This hybridoma was deposited in the National Collection of Cultures of Microorganisms of the Pasteur Institute, (address CNCM, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15), on February 14, 2024, under the number CNCM 1-6035.

[0182] After production of larger volumes of culture supernatants and purification of the antibodies by protein G affinity chromatography, further characterization was then undertaken. Their kinetic parameters (affinity, reflected by the measurement of KD, association and dissociation rates (respectively ka and kd)) for ricin D and ricin E were measured by bi-layer interferometry (BLI) (Table 5 and Figure 2). The affinity measurement by bi-layer interferometry consists of immobilizing the antibodies at a fixed concentration on biosensors containing optical fiber and traversed by a beam of white light. These biosensors will then immerse in different concentrations of ricin (D or E). The association or dissociation of molecules will modify the thickness of the bio-layer on the surface of these sensors and change the way in which the incident white light is reflected inside the sensor. The spectral shift of the interferences created (in nm on the graphs in Figure 2) makes it possible to measure the kinetics of interaction between the molecules.

[0183] [Tables5] Ricin D Ricin E Ko(M) Ml / Ms) Ml / s) MM) ta(l / Ms) ta(1 / s) ERAS <1.0E-12 4.72E+05 <1.0E-07 <1.0E-12 3.35E+05 <1.0E-07

[0184] Table 5: Measurements of the kinetic parameters of the murine ERA5 antibody by BLI for the two isoforms of ricin.

[0185] In vitro neutralization performance (murine antibody)

[0186] The capacity of ERA5 to neutralize ricin was then evaluated with three cell models: Jurkat cells (immortalized cell line of human CD4 T lymphocytes), A549 cells (human type II alveolar lung epithelium cells) and Vero cells (green monkey kidney epithelial cells).

[0187] Ricin at 10 CD50 (CD50 previously determined for each cell type, Table 6) is incubated for 1 h at 37°C with different concentrations of antibodies (100 pg / mL, 667 nM, then diluted by thirds over a maximum of 12 points). Following this incubation, the cells are added at a rate of 1000 cells per well. After 72 h at 37°C, 5% CO2 and 90% humidity, cell viability was assessed by measuring residual ATP (CellTiterGlo® luminescence Cell Viability Assay, #G7570, Promega). LTC5o is determined as the concentration of antibodies required to have 50% cell viability in the presence of 10 CD50 of ricin.

[0188] In the same cell test, the viability of Jurkat cells was measured after contact with equimolar ricin D+E and several different anti-ricin antibodies. More specifically, the viabilities were measured in the presence of the ERA5 antibody of the invention or in the presence of different antibodies of the prior art [6] (antibodies RB34, RB37 and RA36, [Fig.l] and Table 6). These results show that the antibody of the invention has a much better capacity to neutralize ricin D+E than the other two antibodies tested.

[0189] [Tableauxô] RB34 RB37 RA36 ERA5 ICS0 (ng / mL) ndnd 2070 142 ± 35

[0190] Table 6: Comparison of IC50 of published antibodies with ERA5, tested on Jurkat cells in the presence of ricin D+E (equimolar) at 10CD50. nd: not determined because RB34 and RB37 only recognize the D isoform of ricin. With these antibodies, the cells do not reach 50% viability in the equimolar presence of ricin D and E.

[0191] In addition, the anti-ricin efficacy of this antibody could be evaluated in different cellular models (Jurkat, A549, Vero):

[0192] [Tables?] Jurkat A549 Vero Jurkat A549 Vero Dose Ricin (10 CDS1}) pg / mL 96 ± 19,797 + 141,449 ±123 5 CL 1.48 ± 0.29 12.26 ± 2.17 6.91 ± 1.89 ICso ERA5 ng / mL 142 ± 35,308 ± 76 352 ± 85 SG 0.944 ± 0.232 2.05 ± 0.51 2.34 ± 0.57

[0193] Table 7: Determination of the 50% cytotoxic dose for each cell type (Jurkat, A549, Vero)

[0194] These results show that the antibody of the invention ERA5 has good neutralization capacities for the three cell types tested ([Fig.3]).

[0195] It was also possible to exhaustively determine the IC50 of ERA5 with respect to different ricin cultivars (purified ricin of each D and E isoform of the R. Carmencita cultivar used as a reference, and ricin from several other cultivars). The results are presented in [Fig.4]. Thus, it could be demonstrated that the ERA5 antibody of the invention neutralizes ricin from seven cultivars in an equivalent manner, despite differences in cytotoxicity. This is the first anti-ricin antibody described to neutralize ricin from different cultivars.

[0196] In vivo performance (murine antibody)

[0197] Several in vivo experiments were carried out in order to evaluate the protective capacity of the antibody of the invention.

[0198] BALB / c mice were intoxicated with 5 times the LD50 of ricin D+E (LD50 = dose necessary to kill 50% of the mice, previously determined at 20 pg / kg), by intranasal route. At different times after intoxication (6, 10, 18, 24 h), the mice were treated by intravenous injection of one or more antibodies at 10 mg / kg (total amount, regardless of the combinations of antibodies administered, 10 mice per treatment group). They were subsequently followed for 21 days where clinical signs and mortality were recorded daily. As evidenced by the results presented in [Fig. 5], the antibody of the invention ERA5 has excellent protective capabilities against ricin poisoning. Moreover, compared to other antibodies of the prior art (RB34), the protection induced by ERA5 is much superior ([Fig. 6]). [Fig. 6] also presents the effect of a combination of ERA5 with RB34. In this experiment, the amount of ERA5 was divided by 2 so that the total amount of antibodies was equal to the amount of an antibody tested alone. The observed survival is however not divided by 2, which shows a potential additive effect between the two antibodies (or a potential saturation linked to the 10 mg / kg dose used for ERA5).

[0199] The surviving mice from these ERA5-induced protection experiments were re-exposed to the same dose of ricin (5 LD50) 10 to 12 months after the first intoxication, without any other associated treatment. All the mice thus re-exposed survived, with less clinical signs (including weight loss) than during their first exposure (for information, see the re-exposure experiment, the results of which are presented in [Fig. 7]).

[0200] Conclusion:

[0201] As with the in vitro neutralization results, it is difficult to compare the results obtained in vivo with those obtained with the antibodies described in the literature. Indeed, the mouse strains used may have been different, as well as the doses of ricin administered and the 50% Lethal Doses (LD50) equivalents. In addition, the original cultivar or isoform are not always specified, the routes of administration of ricin and / or the antibody may vary, as may the quantities of antibodies. Finally, it is common for experiments to be stopped too early, which is favorable in terms of survival rate, when the animals are still in an unstabilized phase and mortality is still observed.

[0202] Although the results are difficult to compare, on average, after four identical repetitions of the experiment, 92% survival was observed in mice exposed to 5LD50 of ricin intranasally (~2 pg per mouse) and treated 6 hours later with the ERA5 antibody at 10 mg / kg iv. Three of these experiments demonstrated a survival rate of 100%. These results were obtained by measuring the survival of the mice for 21 days, a longer duration than that analyzed in the publications (see Table 2), and less favorable in terms of survival (it often happens that the experiments are stopped after 6 to 14 days in the publications, even though animals still die beyond 14 days - which favors the survival data presented).

[0203] Survival rates of 50% and 35% were also observed with ERA5 (10 mg / kg) injected iv 18h and 24h respectively after exposure to 5LD50 of ricin. These survival rates are higher than any published to date with monoclonal antibodies and for such a wide therapeutic window.

[0204] Active and prolonged immunity induced by passive immunotherapy

[0205] The surviving mice from these ERA5-induced protection experiments were re-exposed to the same dose of ricin (5 LD50) 10 to 12 months after the first intoxication, without any associated treatment. The survival rate of mice in these late re-exposure experiments was 100%, regardless of the time interval between ricin exposure and the administration of the antibody(ies) in the first experiment ([Fig.7]). In addition, a less severe clinical outcome at the second exposure could be observed. In summary, the mice appear to be physiologically less affected by the second intoxication, and long-term active immunity to ricin was therefore induced after treatment of the intoxication with our antibodies.

[0206] Few publications address this type of effect concerning ricin, and never in such a long term, in a murine model. Nevertheless, WG Hu and colleagues report prolonged active immunity 5 months after the first exposure. NJ Mantis and colleagues have shown active immunity induced by the administration of immune complexes of their antibodies with ricin by intranasal or intraperitoneal route in their murine model. This immunity was only demonstrated up to 3 months after the administration of the immune complexes, by exposing these mice to 5 LD50 (1 pg) of uncomplexed ricin 90 days later and evaluating their survival as well as by regular monitoring of anti-ricin antibody titers in the serum of these mice during the 3 months.In this paper, it was also demonstrated that low-dose ricin injection alone was not sufficient to induce prolonged immunity, and that this immunity did depend on the antibodies used for complex formation. These observations suggest that after immediate protection against ricin poisoning, long-term active immunity may also be possible in humans.

[0207] Conclusion

[0208] In the (very unlikely) hypothesis of a second re-exposure to ricin of people who have been exposed and treated with ERA5 a first time, these people would very probably present immune protection to this second exposure. These data also suggest that antibody (ERA5) / ricin immune complexes, administered to people, would allow long-term protection of the vaccine type.

[0209] Murine version ERA5

[0210] The variable heavy (VH) and light (VL) chains of the murine antibody were sequenced by SANGER sequencing: Murine ERA5 VL (highlighted CDRs)

[0211] DIQMTQTTSSLS ASLGDRVTISCRASQDISNYLNWYQQKPDGT VKLLIYYTS RLHSGVPSRFSGSGSGTEYSLTISNLEPEDIATYYCQQYSKRLPTFGGGTKLEIK = SEQ ID NO: 7 Murine VH ERA5 (highlighted CDRs)

[0212] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSDYYWNWIRQFPGNKLEWMGY ISYDGSNNYNPSLNNRISITRDVIKNQFFLKLNSVTTEDTATYYC ARDAHYSNYFDFWGOGTTLTVSS = SEQ ID NO: 8

[0213] Table of CDRs of the (murine) antibodies of the invention

[0214] [Tables8] SEQ ID NO: Amino acid sequence CDR1 Vl 1 QDISNY CDR2 Vl 2 YTS CDR3 Vl 3 QQYSKRLPT CDR1 Vh 4 GYSITSDYY CDR2 Vh 5 ISYDGSN CDR3 Vh 6 ARDAHYSNYFDF

[0215] Table 8: CDRs of the (murine) antibodies of the invention

[0216] Chimeric version of ERA5

[0217] A murine / human chimeric form was recombinantly produced in ExpiCHO cells. This version of ERA5 has murine variable parts and constant CH1, CH2, CH3 fragments of human antibodies. The neutralization capacity was not impacted by this modification ([Fig.8]).

[0218] Humanization of the ERA5 antibody

[0219] After discovering the excellent protection induced by the ERA5 antibody against ricin poisoning, a first humanized sequence (vl) of the variable fragments of ERA5 was produced and tested ([Fig.8]).

[0220] Humanized sequences (mutations in bold underlined) vl VL ERA5 humanized vl [0221 ] DIQMTQSPSSLS ASVGDRVTITCRASQDISNYLNWYQQKPGKAP KLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKRLPTFG QGTKVEIK = SEQ ID NO: 9

[0222] OVOLOESGPGLVKPSOTLSLTCSVSGYSITSDYYWNWIROPPGKG LEWIGYISYDGSNNYNPSLNNRISISVDTSKNOFSLKLNSVTAADTAV YYCARDAHYSNYFDFWGQGTLVTVSS = SEQ ID NO: 10

[0223] This first humanized form, produced recombinantly, neutralizes ricin in vitro but with an IC50 9 times greater than its murine parent ([Fig.8]).

[0224] In order to overcome this loss of neutralization, affinity maturation by molecular engineering was carried out on the humanized version of ERA5.

[0225] The Fab fragment of this humanized form as well as the original murine Fab fragment were expressed on the surface of yeasts in YSD and a combinatorial analysis was carried out by flow cytometry. Five mutated positions of the humanized form were replaced, one by one, by the murine amino acid in order to determine the essential positions to regain the original affinity (see Table 9). The clones allowing to have an affinity equivalent to the murine form were sorted then sequenced by NGS.

[0226] [Tables9] Amino acid position in the VH sequence of ERA5: Murine Humanized 25 71 72 74 75 TTRV 1 SSVTS

[0227] Table 9: Murine and human amino acids, and their positions in the ERA5 sequence, which were varied to effect affinity maturation.

[0228] Among all the sorted yeasts that were able to regain the original binding affinity to ricin, the most frequent population (19% of the total) expresses the murine T and R amino acids at positions 71 and 72 of the ERA5 VH sequence.

[0229] Two new humanized versions were therefore produced: • A “humanized ERA5 v2” antibody whose chain sequence is the same as the humanized ERA5 v1 sequence, but with two substitutions in the variable heavy chain: S71T and V72R. • A bispecific “ERA5-E5” antibody, which is a bispecific version where VHH E5 (having the sequence SEQ ID NO: 12; public sequence as described in Rudolph et al., 2014 (

[11] , https: / / doi.org / 10.1016 / j.jmb.2014.05.026)), and whose structure is described in the Protein Data Bank (PDB) database under the number 4LGP (https: / / www.rcsb.org / structure / 4LGP)) has been grafted onto the heavy chain in the C-terminal position of humanized ERA5 v2.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0230] Human sequences (mutations in soul) v2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> [0231 ] DIQMTQSPSSLS ASVGDRVTITCRASQDISNYLNWYQQKPGKAP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> KLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKRLPTFG QGTKVEIK = SEQ ID NO : 9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0232] VH ERA5 humanity v2 (different from S71T and V72R)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0233] OVOLOESGPGLVKPSOTLSLTCSVSSGYSITSDYYWNWIROPPGKG LEWIGYISYDGSNNYNPSLNNRISITRDTSKNOFSLKLNSVTAADTAV YYCARDAHYSNYFDFWGQGTLVTVSS = SEQ ID NO : 11<h2 style=";text-align:left;direction:ltr">

[0234] The IC50 of all antibodies produced in this work were tested on Jurkat cells contacted with equimolar ricin D+E at 10 CD50. The antibodies (at different concentrations) were pre-incubated for 1 h with equimolar ricin D+E (fixed concentration 10 CD50) and then the Jurkat cells were added (1000 cells / condition). The incubation lasted 72 h and the ATP produced was measured by luminescent reaction (then converted into viability compared to the controls without ricin).

[0235] If we compare the IC5o of the humanized antibody vl with the IC5o of the murine antibody ERA5, we see that this first value is 9 times higher than the second. Thus, we can say that the neutralization capacity of the humanized antibody vl is 9 times lower than that of the murine antibody ERA5 described above. ERA5 murine ERA5 hum vl IC50 (nM) 0.610 5.307 IC50 (pg / mL) 0.092 0.796 ERA5 murine ERA5 hum v2 ERA5-E5 IC50 (nM) 0.448 1.841 0.012 IC50 (pg / mL) 0.067 0.276 0.002

[0236] Furthermore, if we compare the IC5o of the humanized antibody v2 with the IC5o of the murine antibody ERA5, we see that this first value is only 4 times higher than the second. Thus, we can say that the neutralization capacity of the humanized antibody v2 is better than that of the humanized antibody v1 (although less than that of the murine antibody ERA5 described above).

[0237] On the other hand, if we compare the IC50 of the humanized antibody v2 coupled to E5 with the IC50 of the murine antibody ERA5, we see that this first value is 37 times lower than the second. Thus, we can say that the neutralization capacity of the bispecific humanized antibody v2 coupled to E5 is much better than that of the murine antibody ERA5 described above, and, a fortiori, that of the monospecific humanized antibodies v1 and v2.

[0238] Conclusion: Humanized version 2 of ERA5 reduced the performance gap observed between version 1 and the murine version without, however, completely regaining neutralizing capacities. Grafting VHH E5 onto humanized ERA5 v2 significantly improved neutralizing capacities, even exceeding those of the murine version. Bibliographic references

[0239] [2] ES Vitetta, JE Smallshaw, and J. Schindler, “Pilot phase IB clinical trial of an alhydrogel-adsorbed recombinant ricin vaccine,” Clin. Vaccine Immunol., vol. 19, no. 10, pp. 1697-1699, Oct. 2012, doi: 10.1128 / CVI.00381-12.

[0240] [3] PR Pittman et al., “Safety and immunogenicity of ricin vaccine, RVEc™, in a Phase 1 clinical trial,” Vaccine, vol. 33, no. 51, pp. 7299-7306, Dec. 2015, doi: 10.1016 / J.VACCINE.2015.10.094.

[0241] [4] R. Falach et al., “Génération of Highly Efficient Equine-Derived Antibodies for Post-Exposure Treatment of Ricin Intoxications by Vaccination with Monomerized Ricin,” Toxins 2018, Vol. 10, Page 466, vol. 10, no. 11, p. 466, Nov. 2018, doi: 10.3390 / TOXINS 10110466.

[0242] [5] R. Falach et al., “Post-Exposure Anti-Ricin Treatment Protects Swine Against Léthal Systemic and Pulmonary Exposures,” Toxins (Basel)., vol. 12, no. 6, Jun. 2020, doi: 10.3390 / TOXINS 12060354.

[0243] [6] M. L. O. Delgado et al., “Ricin Antibodies’ Neutralizing Capacity against Different Ricin Isoforms and Cultivars,” Toxins 2021, Vol. 13, Page 100, vol. 13, no. 2, p. 100, Jan. 2021, doi: 10.3390 / TOXINS 13020100.

[0244] [7] J. Prigent et al., “Neutralising Antibodies against Ricin Toxin,” PLoS One, vol. 6, no. 5, p. 20166, 2011, doi: 10.1371 / JOURNAL.PONE.0020166.

[0245] [8] W. G. Hu, J. Yin, D. Chau, L. M. Negrych, and J. W. Cherwonogrodzky, “Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody,” PLoS One, vol. 7, no. 9, Sep. 2012, doi: 10.1371 / journal.pone.0045595.

[0246] [9] T. Pelât, M. Hust, M. Haie, M. P. Lefranc, S. Dübel, and P. Thullier, “Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity,” BMC Biotechnol., vol. 9, no. 1, pp. 1-13, Jun. 2009, doi: 10.1186 / 1472-6750-9-60 / TABLES / 4.

[0247]

[10] C. Herrera, D. J. Vance, L. E. Eisele, C. B. Shoemaker, N. J. Mantis, and E. R. Goldman, “Differential Neutralizing Activities of a Single Domain Camelid Antibody (V H H) Spécifie for Ricin Toxin’s Binding Subunit (RTB),” 2014, doi: 10.1371 / journal.pone.0099788.

[0248]

[11] M. J. Rudolph et al., “Crystal structures of ricin toxin’s enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies,” J. Mol. Biol., vol. 426, no. 17, pp. 3057-3068, 2014, doi: 10.1016 / j.jmb.2014.05.026.

[0249]

[12] M. J. Rudolph et al., “Structural Analysis of Toxin-Neutralizing, Single-Domain Antibodies that Bridge Ricin’s A-B Subunit Interface,” J. Mol. Biol., vol. 433, no. 15, Jul. 2021, doi: 10.1016 / J.JMB.2021.167086.

[0250]

[13] C. Herrera, T. I. Klokk, R. Cole, K. Sandvig, and N. J. Mantis, “A Bispecific Antibody Promûtes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking,” PLoS One, vol. 11, no. 6, p. 156893, 2016, doi: 10.1371 / joumal.pone.0156893.

[0251]

[14] J. M. O’hara, L. M. Neal, E. A. Mccarthy, J. A. Kasten-Jolly, R. N. Brey lii, and N. J. Mantis, “Folding domains within the ricin toxin A subunit as targets of protective antibodies,” Vaccine, vol. 28, pp. 7035-7046, 2010, doi: 10.1016 / j.vaccine.2010.08.020.

[0252]

[15] J. Prigent et al., “Neutralising antibodies against ricin toxin,” PLoS One, vol. 6, no. 5, 2011, doi: 10.1371 / JOURNAL.PONE.0020166.

[0253]

[16] T. Noy-Porat et al., “Isolation of anti-ricin protective antibodies exhibiting high affinity from immunized-human primates,” Toxins (Basel)., vol. 8, no. 3, Mar. 2016, doi: 10.3390 / toxins8030064.

[0254]

[17] N. Dong et al., “Monoclonal antibody, mAb 4C13, an effective detoxicant antibody against ricin poisoning,” Vaccine, vol. 33, no. 32, pp. 3836-3842, Jul. 2015, doi: 10.1016 / J.VACCINE.2015.06.096.

[0255]

[18] C. C. Hu, J. Yin, D. Chau, J. W. Cherwonogrodzky, and W.-G. Hu, “Active Immunity Induced by Passive IgG Post-Exposure Protection against Ricin,” Toxins (Basel)., vol. 6, pp. 380-393, 2014, doi: 10.3390 / toxins6010380.

[0256]

[19] S. H. Pincus, A. Das, K. Song, G. A. Maresh, M. Corti, and J. Berry, “Rôle of Fc in antibody-mediated protection from ricin toxin,” Toxins (Basel)., vol. 6, no. 5, pp. 1512-1525, May 2014, doi: 10.3390 / toxins6051512.

[0257]

[20] E. K. Sully et al., “Chimeric plantibody passively protects mice against aerosolized ricin challenge,” Clin. Vaccine Immunol., vol. 21, no. 5, pp. 777-782, 2014, doi: 10.1128 / CVI.00003-14.

[0258]

[21] J. M. O’Hara, K. Whaley, M. Pauly, L. Zeitlin, and N. J. Mantis, “Plant-based expression of a partially humanized neutralizing monoclonal IgG directed against an immunodominant epitope on the ricin toxin A subunit,” Vaccine, vol. 30, no. 7, pp. 1239-1243, Feb. 2012, doi: 10.1016 / J.VACCINE.2011.12.058.

[0259]

[22] L. E. Tolman et al., “Durable Immunity to Ricin Toxin Elicited by Intranasally Administered Monoclonal Antibody-Based Immune Complexes,” ImmunoHorizons, vol. 6, no. 6, pp. 324-333, Jun. 2022, doi: 10.4049 / IMMUNOHORIZONS.2100105.

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] FR2922212 BioEssays, 8:74, 1988 Jones et al., Nature, 321:522-525, 1986 Verhoeyen et al., Science, 239:1534-1536, 1988 Riechmann et al., Nature, 332:323-327, 1988 Singer et al., J. Immun. 150:2844-2857, 1992 Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992 Bebbington et al., Bio / Technology, 10:169-175, 1992 EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647, US 5,530,101, US 6,180,370, US 5,585,089, US 5,693,761.

Claims

Claims

1. An isolated and purified antibody or antibody fragment capable of recognizing and neutralizing ricin isoforms D and E, comprising: a. a light chain comprising the CDRs having the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or having a sequence identity of at least 80% with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and b. a heavy chain comprising the CDRs having the sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or having a sequence identity of at least 80% with SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

2. Antibody or antibody fragment according to claim 1, characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 7, or an amino acid sequence having at least 80% identity with SEQ ID NO:

7.

3. Antibody or antibody fragment according to claim 1 or 2, characterized in that its heavy chain contains the variable domain VH of sequence SEQ ID NO: 8, or an amino acid sequence having at least 80% identity with SEQ ID NO:

8.

4. Antibody or antibody fragment according to any one of claims 1 to 3, characterized in that its affinity with ricin D and with ricin E of the cultivar R. carmencita is less than 10 12 measured by bi-layer interferometry (BLI).

5. Antibody or antibody fragment according to claim 4, said antibody being humanized, or human, characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 9, or an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and in that its heavy chain contains the variable domain VH of sequence SEQ ID NO: 11, or an amino acid sequence having at least 80% identity with SEQ ID NO:

11.

6. Antibody or antibody fragment according to claim 4, said antibody being humanized, or human, characterized in that its light chain contains the variable domain VL of sequence SEQ ID NO: 9, or an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and in that its heavy chain contains the domain VH variable of sequence SEQ ID NO: 10, or an amino acid sequence having at least 80% identity with SEQ ID NO:

10.

7. An antibody or antibody fragment according to any one of claims 1 to 6, said antibody being bispecific and coupled to the VHH E5 antibody.

8. A pharmaceutical composition comprising the antibody or antibody fragment as described in claims 1 to 7, and a pharmaceutically acceptable excipient.

9. A pharmaceutical composition according to claim 8, for use in protecting against or reducing the effects of ricin poisoning.

10. An isolated nucleic acid, chosen from: a) a nucleic acid, DNA or RNA, coding for the antibody or one of its fragments as defined in claims 1 to 7; b) a nucleic acid whose sequence has a percentage identity of at least 80% after optimal alignment with one of the sequences a).

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