IT201900011676A1 - Human recombinant antibody against the LAG3 membrane receptor, its medical and diagnostic uses. - Google Patents

Description

HUMAN RECOMBINANT ANTIBODY AGAINST THE RECEPTOR OF

LAG3 MEMBRANE, ITS MEDICAL AND DIAGNOSTIC USES

The present invention relates to a recombinant human antibody directed against the LAG3 membrane receptor, and its related medical and diagnostic uses. In particular, the present invention relates to an anti-LAG3 single-chain variable fragment (scFv) derived from the 'phage display' technology which targets dysfunctional CD8 lymphocytes, and which therefore can be advantageously used in the treatment of diseases characterized by a lymphocyte dysfunction in which LAG3 is involved, such as some tumors, and in the in vitro diagnosis of these diseases.

It is known that in chronic diseases, including some cancers, possibly due to persistent exposure of the antigen, there is an altered expression of molecules with regulatory activity on the immune system (called immune checkpoints, IC), including LAG3, with the consequent acquisition of a dysfunctional phenotype of specific lymphocytes, which progressively lose their immunoprotective properties [1]. The understanding of this tumor "escape" mechanism has paved the way for new immunotherapeutic strategies based on the use of monoclonal antibodies capable of binding certain inhibitory receptors (IRs) - belonging to a group of IC molecules over-expressed on tumor lymphocytes - specific - and able to interfere with their inhibitory signals in order to restore a clinically significant immune response against tumor cells [1, 2].

This therapeutic design has generated considerable enthusiasm in the field of oncology since it has provided persistent clinical benefits for a significant number of patients with advanced cancer. Unfortunately, however, despite the surprising and persistent results obtained in an important number of patients with the "pioneering" antibodies of this new class of biological pharmaceutical products - directed against the CTLA-4 and PD-1 receptors - many patients continue to be unresponsive. to current therapies based on 'IC Blockers' (ICB). Furthermore, immune activation can be associated with immune-related adverse events affecting various organs, including skin, intestines, heart, lungs and bones [3]. The reasons must be sought in the complexity of the immune system responses, as well as in the differences between the different clinical cases, in the genetic background of each patient and in the involvement of other inhibitory molecules involved in the acquisition of a dysfunctional condition of the immune system [4- 6,7, 8].

IRs guarantee tolerance towards 'self' and modulate duration and amplitude of physiological immune responses, but it is now clear that tumors could hijack some regulatory pathways as the main mechanism of immune resistance, in particular in CD8 T lymphocytes specific for tumor antigens. . In this context, specific monoclonal antibodies (mAbs) constitute powerful therapeutic agents due to their ability to selectively bind IRs thus interfering with the regulatory pathways they control, restoring an effective immune response [9]. Meanwhile, awareness is emerging that several IRs may be involved in lymphocyte dysfunction [4-6]. It follows that using a combinatorial approach aimed at the different IRs expressed could increase the quality of the results in the oncology clinic [10,11]. A first confirmation of this hypothesis came from the fact that the combination of two 'IR blockers', namely anti-PD1 and anti-CTLA4 mAb, obtained a higher response rate than that observed with single antibody treatments.

Therefore, nowadays the focus is on evaluating new immune checkpoint inhibitors and their combinations to increase their efficacy and at the same time reduce toxicity [12].

Lymphocyte activate gene (LAG) 3 (also known as CD223) is a type I 498 aa transmembrane protein that acts as an inhibitory immune receptor (IR). LAG3 [13], together with other molecules already used for clinical purposes (such as PD1 and CTLA-4), is a membrane receptor belonging to the family of "immune control points" (ICP), proteins usually involved in the regulation of immune responses [14, 15]. LAG3 shows about 20% identity with CD4 [13] and, like CD4, binds MHC class II molecules, albeit to a distinct site and with a higher affinity [16,17]. LAG3 is not expressed by resting T cells, but is instead upregulated on activated T lymphocytes (both the CD4 and CD8 subpopulations), on B lymphocytes, 'natural killer' cells, and plasmacytoid dendritic cells (DC). In activated lymphocytes, LAG3 expression is involved in the negative control of cell activation / proliferation to ensure the modulation and control of immune responses.

In addition to MHC class II molecules, LAG3 has been shown to bind Galectin-3 [18, 19], LSECtin [20] and, in the central nervous system, α-synuclein [21]. Activation of LAG3 leads to a negative regulation of T cell activation. However, the signaling mechanism of LAG3 is still largely unknown. A couple of still widely conserved unique domains, namely EP [22] and KIEELE [23], appear to be important for the transmission of intracellular inhibitory signals. Furthermore, LAG3 shows a bidirectional signaling activity, as deduced from the modulation of the activation / maturation of DC observed following the interaction with lymphocytes expressing LAG3 [24, 25].

LAG3 has been identified as one of the major inhibitory receptors (IRs) involved in T-cell dysfunction typically occurring in oncological diseases [26]. In light of the dysregulated expression of LAG3 in tumor-infiltrating lymphocytes, specific anti-LAG3 mAbs are currently being investigated in an attempt to remedy cancer-associated immunosuppression. Along with other IRs, namely CTLA-4 and PD-1, LAG3 is now considered an important target of the so-called anti-tumor strategy based on blocking immune checkpoints (ICB) by administering mAbs versus IR [1].

In particular, in the oncological context, and more specifically in solid tumors, LAG3 is up-regulated on tumor-infiltrated lymphocytes (TIL) and the blocking of LAG3-triggered signals obtained with specific antibodies can improve the responses of antitumor T cells. It has been shown, in both mice and humans, that the simultaneous blocking of PD1 and LAG3 is even more efficient than interference on only the two molecules [13, 27]. Furthermore, it has been shown that previous exposure to anti-PD1 antibodies can enhance LAG3 expression, promoting the dysfunctional T cell phenotype, and possibly leading to resistance to anti-PD1 drugs.

The interest in the identification and characterization of new ICBs is constantly growing as evidenced by the various clinical studies and by the rather recent publication of works describing the attempts for the isolation of new anti-IR antibodies (Abs), including antibodies anti-LAG3.

Monoclonal antibodies (mAb) are molecules widely used in therapy, diagnostics and biotechnology. In some cases, the efficacy of whole antibody molecules may be limited for some applications due to their large hetero-tetrameric structure. For this reason, the complex structure of Abs can be reduced in several ways without affecting their effectiveness. Among these modalities, the single-chain variable fragment (scFv) is one of the simplest immunoglobulins that maintain the activity of binding with the antigen unaltered [28]. This molecule is formed by the variable regions of both heavy and light chains joined by a peptide linker. Although scFvs have the significant advantage that they can be expressed from a single reading frame, their low stability and short pharmacokinetics may limit their applications. The fusion of an scFv with the CH2 and CH3 domains of the immunoglobulin (Ig) Fc region can overcome these drawbacks [29].

To avoid undesirable immune responses, human Abs are the most suitable reagents for therapeutic purposes. Human scFvs can be isolated through the well-established 'phage display' technique. A study by Everett and his collaborators (coll.) Reports the isolation of Abs anti-LAG3 able to interact with both human and murine LAG3 through an additional engineered binding domain within the CH3 region of the Fc portion [30 ]. The in vitro activity of these novel antibody constructs has been shown to increase IL-2 release from stimulated PBMCs. In particular, Everett describes the isolation of molecules capable of binding LAG3, isolated from a library generated on an Fc-type 'scaffold', ie by introducing randomized sequences in the CH3 region of the constant portion of a human IgG1. These reagents (called FcAb) were therefore designed to produce bispecific recombinant molecules with a structure similar to that of a traditional antibody but with three binding sites, one of which at the end of the constant Fc portion. The functionality was evaluated in terms of the interference-related effect of the LAG3-MHCII interaction, in particular through an in vitro assay based on the quantization of IL-2 (with ELISA kit) in the co-culture surpernatants of a lymphocyte line. T CD4 - Do11.10 OVA (transduced to obtain an overexpression of LAG3) and an A20 lymphoma line expressing (endogenously) MHCII. In fact, the LAG3-MHCII interaction in this model involves a reduced expression of IL-2, which instead is improved in the presence of molecules that interfere with this interaction. Furthermore, Sasso and his colleagues [31] described a procedure for Abs selection based on the binding of phagemid particles on activated human lymphocytes to generate a repertoire of scFvs against various IRs including LAG3. In particular, Sasso et al. describe the isolation of human scFv antibodies from a synthetic library (Andrew D. Griffiths et al.), then reconstructed into a whole igG4 antibody isotype.

After reconstitution into human IgG4, its biological activities were assessed by cytometric and ELISA-based lymphocyte proliferation assays to detect cytokines. In particular, the antibody binding activity and its functional properties were evaluated on PBMC activated with PHA, respectively by flow cytometric analysis and ELISA for monitoring IL-2 and INF-γ released in the supernatant of treated cells in different intervals. of time.

Other antibodies that bind to LAG-3 are described in US2018066054, US2018371087.

It is also known that the success of ICB-based immunotherapies will depend on the combination of several optional treatments. In a rapidly evolving research context, the generation of a repertoire of different immunomodulatory Ab-based constructs could play an essential role in the identification of effective therapeutic agents, and LAG3 certainly represents one of the most interesting emerging targets. The great interest in LAG3 has been fueled by its strong expression in dysfunctional T cells associated with many human cancers [27, 32, 33].

Furthermore, there is strong evidence that the altered over-expression of LAG3 could mediate a mechanism for a tumor escape route from PD1-based therapy, whose acquired resistance could be overcome precisely by the combined treatment with anti-ICB LAG3 [34 -37]. Furthermore, LAG3, together with TIGIT and Tim-3, is expected to have a better safety profile in the clinic than the combinations CTLA4 and PD1 [13, 12]. In addition, LAG3 blockade inhibits the immuno-suppressive activity of Treg cells both in vitro and in vivo in an autoimmune pulmonary vasculitis model [38], thus fueling the intriguing hypothesis that a similar beneficial effect may also be obtained in the tumor microenvironment. For all these reasons, LAG3-specific ICBs are currently being evaluated in phase I and II clinical trials in a variety of solid and haematological malignancies both alone and in combination with PD1.

However, although there are some anti-Lag antibodies in clinical trials, no anti-Lag3 antibodies to date have been approved for clinical use; Lag3 is a very complex molecule and is probably able to interact with different ligands, so the therapeutic effect of different antibodies directed against different epitopes can be equally varied, in different patients and in different pathological contexts. Therefore, having a panel of LAG3 antibodies would mean increasing the chances of success for therapies based on the interference of this ICP and / or expanding the therapeutic options in clinical practice.

In light of the above, it is therefore evident the need to develop new anti-LAG3 antibodies, which overcome the limitations of the already known anti-LAG3 antibodies.

According to the present invention, a new recombinant human antibody (Ab) against a conformational epitope of LAG3 has now been identified. In particular, a single-chain variable fragment human antibody (scFv) was isolated in vitro by 'phage display' technology using the recombinant antigen as bait. The antibody described in the present invention is capable of specifically intercepting the LAG-3 receptor and can be advantageously used both in the diagnosis for the preselection of patients to be treated and in therapy for the treatment of selected patients. The effect of the antibody of the present invention is focused on a particular lymphocyte population. Data obtained with an in vitro model demonstrate the ability of the antibody to act selectively on specific CD8 cells. The clinical importance of CD8 lymphocytes infiltrated into the tumor mass (TIL) is known precisely in the context of immunotherapy based on immune checkpoint inhibitors. Therefore, the antibody described in the present invention expands the therapeutic options available and / or could enhance those already approved by the competent authorities, with the ultimate goal of improving the efficacy of the treatments and increasing the pool of patients who can benefit from these innovative immunotherapy strategies.

In particular, the antibody of the present invention was selected from a completely human 'naive' phage library by means of an in vitro 'bio-panning' strategy.

This scFv (referred to as F7) has been characterized in terms of binding specificity both on the recombinant antigen and on human cells expressing LAG3. It was then reconstructed in a pre-optimized IgG format for clinical use and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Furthermore, according to the present invention, the activity of anti-LAG3 scFvF7 was analyzed using two different antigenspecific CD8 T lymphocyte clones as target cells. The reconstituted anti-LAG3 F7 Ab effectively binds the cell membrane of both cell clones after activation with the specific peptide. Importantly, it has been observed that treatment with Ab causes a significant increase in peptide-dependent cell activation, measured in terms of IFN-γ release both by ELISA and by ELISPOT assays. Therefore, after reconstruction with the Fc domain, this Ab maintains its binding activity, and significantly improves the activation of antigen-specific CD8 + T lymphocytes.

The completely new molecule object of the present invention targets a conformational epitope of the LAG3 antigen and has been isolated from a 'naïve' phage library (IORISS1) [39], completely human and different from those used for anti- Existing LAG3. As such, it is identified by a unique gene and amino acid sequence, never isolated and previously published.

The anti-LAG3 antibody according to the present invention was developed in a recombinant format of the type (scFv) 2-Fc (= CH2 / CH3) pre-optimized for clinical use, which is different from those described in the papers published by Sasso et al. and Everett et al .. Furthermore, the antibody of the present invention was designed to maintain an adequate serum half-life and to eliminate side effects dependent on the constant Fc domain. The obtained bivalent construct preserves the binding efficiency, as shown in flow cytometry both on cells transfected with LAG3 and on human lymphocytes activated with PHA.

Unlike the already known anti-LAG3 antibodies, the experimental results show that the LAG3 antibody described in the present invention is able to strongly increase the antigen-specific activation of CD8 T lymphocytes, using a particular in vitro model that allows to highlight the effect of the antibody on this particular cellular subpopulation.

This model is in fact based on human CD8 T lymphocyte clones, activated with "B LCL cells having HLA compatible", previously "loaded" with the corresponding specific peptides, namely a tumor peptide (Mart-1) and a viral peptide (Nef / HIV) , where the presence of the antibody determines a strong increase in the expression of INFγ as shown in ELISA and in ELISPOT assays.

Despite the lack of "standardized" methods for functional assessment and adequate comparison of ICP blocking agents, the model used in the present invention allows to precisely target the ability of the scFvF7 antibody construct to enhance activation of CD8 lymphocytes stimulated with their specific peptides 'presented' in a way that can be superimposed on a normal physiological process.

This aspect is very important considering the known clinical relevance of specific CD8 lymphocytes, in particular those that infiltrate the tumor (where LAG3 expression is often dysregulated), in order to reorient the immune system towards an effective response. Furthermore, the specificity requirements, the molecular structure, designed for clinical use, together with its functional properties, make the scFvF7 antibody a privileged tool for different types of applications.

The anti-LAG3 antibody of the present invention can certainly be used to monitor the expression of LAG-3 levels on human cells. Therefore - together with other reagents - it can be used in diagnostic protocols that aim to identify patients with the highest chance of responding effectively to specific ICP blocker therapy, in line with the growing need for personalized medicine [7, 8 , 10, 11]. Furthermore, the scFvF7 antibody meets all the requirements to be an immunomodulatory drug by virtue of its completely human structure - therefore little or no immunogenic - and its ability to interfere with inhibitory signals mediated by LAG-3, the expression of which is often dysregulated on tumor specific CD8 lymphocytes or virus - tumor specific in chronic diseases.

The specific object of the present invention is therefore a human recombinant antibody sequence against a conformational epitope of LAG3 comprising or consisting of

a VH sequence comprising the CDR sequence AKDQDGGKKTDAFDI (SEQ ID NO: 3), said VH sequence optionally further comprising the following CDR sequences:

ISAGGTGT (SEQ ID NO: 2), or

ISAGGTGT (SEQ ID NO: 2) and GFTFSSYA (SEQ ID NO: 1), or GFTFSSYA (SEQ ID NO: 1)

And

a VL sequence comprising the CDR sequence QQTGYYPLT (SEQ ID NO: 5), said VL sequence optionally comprising the following CDR sequences:

AAS, or

AAS and QGINYY (SEQ ID NO: 4), or

QGINYY (SEQ ID NO: 4).

Said VH and VL sequences are coupled to reproduce the binding site of the antibody by methods which are known to a person skilled in the art. According to an embodiment of the present invention, the human recombinant antibody sequence may be in the form of a human single chain variable antibody fragment (scFv) sequence. In this case, said VH and VL sequences are connected by a 'linker' designed to join said sequences.

However, other methods are known for the association of VH and VL sequences; for example VH and VL can be genetically fused respectively to the constant domains of heavy and light chains, whose natural interaction favors the approach of the variable regions, crucial for the formation of the binding site.

More generally, the modular architecture of antibody domains has been exploited to create an increasing number of alternative formats, including artificial structures in which VH and VL pairing is achieved by genetic fusion with proteins (or protein fragments). prone to association / multimerization.

According to an embodiment of the present invention, said VH sequence may comprise or consist of MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCAKDQDGTM: NOGTVDI (QGTVDI)

said VL sequence may include or consist of DIVMTQSPSLLSASVGDRVTITCRASQGINYYLAWYQQKPGKAPKLLIYAASTLQ SGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQTGYYPLTFGPGTKVDI

(SEQ ID NO: 7), said VL sequence optionally also comprising a restriction site at the C terminal end, such as KAAA (SEQ ID NO: 8).

The above mentioned linker can be for example GGGGSGGGGSGGGGS (SEQ ID NO: 9).

Secondo una forma di realizzazione, la sequenza anticorpale può comprendere o consistere in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDI (SEQ ID NO: 10), facoltativamente comprendente ulteriormente un sito di restrizione all’estremità C terminale, come KAAA (SEQ ID NO: 8).

Secondo un'ulteriore forma di realizzazione della presente invenzione, la sequenza anticorpale può comprendere o consistere in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAADDDSDDDYKDDDDQHHHHHH

(SEQID NO: 11).

The antibody sequence may further comprise human constant immunoglobulin domains, such as CH2-CH3 or CH3 where optionally the CH2 domain comprises LALA mutations.

For example, the CH2 sequence can include or consist of APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

G (SEQ ID NO: 12), the CH3 sequence may comprise or consist of QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMKSRWQGNVFSCSVMKSRWGHNLSPKSL

(SEQ ID NO: 13) and the CH2 sequence with LALA mutations may comprise or consist of APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

G (SEQ ID NO: 14).

Said constant human immunoglobulin domains can be linked to the VL sequence by a hinge, such as EPKSCDKTHTCPPCP (SEQ ID NO: 15).

Secondo una forma di realizzazione della presente invenzione, la sequenza anticorpale può comprendere o consistere in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAAASEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (ID SEQ NO: 16).

Secondo un'ulteriore forma di realizzazione del presente invenzione, la sequenza anticorpale può comprendere o consistere in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAAASEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (ID SEQ NO: 17).

The antibody sequence according to the present invention can be in bivalent form. In particular, the bivalent form is obtained after the translation of proteins as an effect of a spontaneous coupling between the constant portions (CH2-CH3 or CH3) of two monovalent molecules.

The present invention also relates to a nucleotide sequence, which encodes the antibody sequence as defined above.

The VH-encoding nucleotide sequence may comprise the CDR nucleotide sequence GCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCTTTTGATATC (ID SEQ NO: 20), said VH-encoding nucleotide sequence may optionally also comprise the following CDR nucleotide sequences:

ATTAGTGCTGGTGGTACTGGCACA (SEQ ID NO: 19), or ATTAGTGCTGGTGGTACTGGCACA (SEQ ID NO: 19) and GGATTCACCTTTTAGCAGCTATGCC (SEQ ID NO: 18), or GGATTCACCTTTAGC (SEQ ID NO: 19) and ID

the nucleotide sequence encoding VL may comprise the nucleotide sequence CDR CAGCAGACTGGTTATTACCCTCTCACT (SEQ ID NO: 22), said nucleotide sequence encoding VL optionally also comprising the following nucleotide sequences CDR:

GCTGCATCC, or

GCTGCATCC and CAGGGCATTAACTATTAT (SEQ ID NO: 21), or

CAGGGCATTAACTATTAT (SEQ ID NO: 21).

According to an embodiment of the present invention, the nucleotide sequence encoding VH can comprise or consist of:

ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (ID SEQ NO: 23).

According to a further embodiment of the present invention, the nucleotide sequence encoding VL can comprise or consist of:

GATATTGTGATGACGCAGTCTCCATCCCTCCTGTCTGCATCTGTAGGAGACAGAG TCACCATCACTTGCCGGGCCAGTCAGGGCATTAACTATTATTTAGCCTGGTATCA GCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAA AGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCA CAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGACTGG TTATTACCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATC (ID SEQ NO: 24), facoltativamente comprendente inoltre una sequenza nucleotidica che codifica un sito di restrizione alla estremità 3’, ad esempio AAAGCGGCCGCA (SEQ ID NO: 25).

The nucleotide sequence encoding the above mentioned linker connecting VH and VL can be: GGTGGAGGTGGATCTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCC (ID SEQ NO: 26).

According to one embodiment, the nucleotide sequence can comprise or consist of:

ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATC (SEQ ID NO: 27), facoltativamente comprendente anche una sequenza nucleotidica codificante un sito di restrizione all'estremità 3’, come AAAGCGGCCGCA (SEQ ID NO: 25).

According to a further embodiment of the present invention, the nucleotide sequence comprising or consisting of:

ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGATGACGATTCTGACGATGACTA CAAGGACGACGACGACCAGCACCATCACCATCACCATTAG (SEQ ID NO: 28).

The antibody nucleotide sequence may further comprise a nucleotide sequence encoding a constant human immunoglobulin domain, such as CH2-CH3 or CH3 where optionally the CH2 domain may comprise LALA mutations. Per esempio, la sequenza nucleotidica codificante per CH2 può comprendono o consistere in GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA GGG (SEQ ID NO: 29), la sequenza nucleotidica codificante per CH3 può comprendere o consistere in CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (ID SEQ NO : 30) and the nucleotide sequence encoding CH2 with LALA mutations may comprise or consist of GCACCTGAAGCTGCTGGGGGGACCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA GGG (SEQ ID NO: 31).

Said nucleotide sequence encoding human constant immunoglobulin domains can be linked with the nucleotide sequence encoding VL from a nucleotide sequence encoding a zipper such as GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA (ID SEQ NO: 32).

According to an embodiment of the present invention, the nucleotide antibody sequence, which comprises CH2-CH3, can comprise or consist of:

ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGCTAGCGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA (SEQ ID NO: 33).

According to a further embodiment of the present invention, the nucleotide antibody sequence, which comprises CH2-CH3, can comprise or consist of:

ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGCTAGCGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCTGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA (SEQ ID NO: 34).

A further object of the present invention is a vector comprising the nucleotide sequence as defined above.

The vector can be selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, or non-viral vectors.

The invention also relates to a cell comprising the vector as defined above, in which said cell can preferably be a eukaryotic cell, more preferably a CHO cell.

The present invention relates to a pharmaceutical composition comprising or consisting of an antibody sequence as defined above, a nucleotide sequence as defined above, a vector as defined above or a cell as defined above, as an active ingredient, together with one or more excipients and / or adjuvants.

The pharmaceutical composition may further comprise at least one other therapeutic active ingredient including antibodies, cytokines, chemical drugs, preferably capable of blocking an immune checkpoint, such as anti-CTLA4, anti-PD1 antibodies.

A further object of the present invention is the antibody sequence as defined above, a nucleotide sequence as defined above, a vector as defined above, a cell as defined above or a pharmaceutical composition as defined above, for use as a medicament.

The present invention also relates to the antibody sequence as defined above, a nucleotide sequence as defined above, a vector as defined above, a cell as defined above or a pharmaceutical composition as defined above, for use in the treatment of a disease characterized by a immune dysfunction in which LAG3 is involved.

Diseases characterized by a malfunction of the lymphocytes in which LAG3 is involved can be selected from a group consisting of human cancers such as NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, solid and haematological tumors, such as melanoma, follicular lymphoma, infectious diseases such as chronic infectious diseases, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection.

An altered expression of LAG-3 has been found in a broad spectrum of human cancers, such as melanoma, NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, follicular lymphoma, squamous cell carcinoma of the head and neck, etc. , and is significantly associated with tumor progression and aggressive clinicopathological features.

Novel anti-Lag-3 antibodies are currently being evaluated in several clinical trials, in many hematological and solid cancers, where they are producing exciting preliminary results especially in combination with other immunecheckpoint (i.e. anti-PD-1) blockers and a good profile safety. These results are of particular interest for a broader exploration of LAG-3 as an alternative immunotherapy target and potential predictive bio-marker.

In infectious diseases, when the immune system fails to eradicate the infection quickly, numerous inhibitory signals are triggered, including those controlled by LAG-3, to curb the response and prevent any immune-mediated damage to the host's tissues, promoting thus a dysfunctional condition of immune responses. Therefore, even in the context of chronic infectious diseases (such as those caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV)), there is evidence to support effective clinical use of anti-LAG3 mAbs [40, 41].

The present invention also relates to a combination of an antibody sequence as defined above, a nucleotide sequence as defined above, a vector as defined above, a cell as defined above or a pharmaceutical composition as defined above, with at least one other active therapeutic substance such as antibodies, cytokines, chemical drugs, preferably blockers of immune checkpoints, such as anti-CTLA4, anti-PD1 antibodies, for sequential or separate use in the treatment of a disease characterized by a lymphocyte dysfunction in which LAG3 is involved.

Diseases characterized by lymphocyte dysfunction in which LAG3 is involved can be selected from the group consisting of human cancers such as NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, solid and hematological tumors, such as melanoma , follicular lymphoma, chronic infectious diseases, such as infections caused by human immunodeficiency virus (HIV), hepatitis B virus and hepatitis C virus (HCV).

Currently, there is strong evidence regarding the synergistic effect of anti-Lag-3 antibodies with PD-1 antibody. However, this is a young field in constant evolution, and other combinations (with other ICP blocking molecules and with other active substances, such as antibodies, cytokines, chemical drugs) could prove effective in the near future.

According to the present invention, the term "separate use" means an administration, at the same time, of the two compounds of the composition according to the invention in distinct pharmaceutical forms.

The term "sequential use" is intended as a subsequent administration of the two compounds of the composition according to the present invention, each of which in a distinct pharmaceutical form.

The present invention also relates to the use of an antibody sequence according to the present invention for the in vitro diagnosis of a disease which is characterized by a lymphocytic dysfunction in which LAG3 is involved.

Diseases characterized by a lymphocyte dysfunction in which LAG3 is involved can be selected from the group consisting of human cancers such as NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, solid and hematological tumors, such as melanoma , follicular lymphoma, chronic infectious diseases, such as infections caused by human immunodeficiency virus (HIV), hepatitis B virus and hepatitis C virus (HCV).

An object of the present invention is a kit for in vitro diagnosis of a disease which is characterized by a lymphocyte dysfunction in which LAG3 is involved, said kit comprising an antibody sequence according to the present invention.

As mentioned above, diseases characterized by a lymphocyte dysfunction in which LAG3 is involved, can be selected from the group consisting of human cancers such as NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, hematological and solid cancers, such as melanoma, follicular lymphoma, chronic infectious diseases, such as infections caused by human immunodeficiency virus (HIV), hepatitis B virus and hepatitis C virus (HCV).

The present invention will now be described in an illustrative, but not limiting way, according to its preferred embodiments, with particular reference to the attached drawings, which show:

Fig. 1. Isolation and 'screening' of anti-LAG3 scFv using the 'Phage display' technology. A. Scheme of affinity-based phage selection for LAG3, starting from the IORISS1 Antibody Library. B. Anti-LAG3 ELISA performed with the supernatants of 90 isolated bacterial colonies after five rounds of panning. The plate was coated with 0.5 µg of antigen for each micro well. The numbers identify each positive clone. O.D .: optical density.

Fig. 2. Nucleotide and amino acid sequences of scFvF7. The entire scFvF7 sequence is shown, including the tags. The CDR1, CDR2 and CDR3 regions of VH and VL are indicated in bold. The linker region is shown underlined (nucleotide sequence 373-417, amino acid sequence 125 - 139. The tag-flag sequence (amino acid sequence 256-263) is also underlined, while the last six amino acid residues represent the tagged region with 6 histidinines , positioned immediately before the stop nucleotide triplet ('TAG').

Fig. 3. Characterization of anti-LAG3 scFvF7. A. SDS-PAGE analysis of 1 and 5 µg of scFvF7. Molecular markers in kilodalton (kDa) are shown on the right. B. ELISA for the detection of LAG3 antigen performed with decreasing doses of purified scFvF7. GO: glucose oxidase. O.D .: optical density. The means of the ± SEM values calculated from the results of three independent assays are shown. C. FACS analysis performed with scFvF7 on human CD4 T lymphocytes not stimulated or stimulated with PHA. Insert: the results of the same test carried out with the mouse 17B4 anti-LAG3 mAb are reported. The data shown are representative of four independent experiments. D. Binding analysis of scFvF7. 0.5 µg of recombinant LAG3 protein and glucose oxidase (GO) were loaded into well replicas on a 12% SDS-PAGE and then transferred to filter paper. Strips obtained from the filter were then incubated with the indicated antibodies. An anti-His 6 mAb was used as a positive control for the recombinant LAG3 protein (which has a 6 histidine tag at its C-terminus). As additional negative controls, two filter strips were incubated with the anti-Flag antibody and the HRP-conjugated anti-mouse antibody, respectively, in order to monitor for any non-specific signals due to the secondary antibodies used to detect the scFv antibodies. The ELISA reported below (performed with the same secondary Abs) is a control for the reactivity of the supernatants containing the scFvs (scFvGO and scFvF7) used in the assay. Arrows indicate relevant signs. Molecular markers in kilodaltons (kDa) are shown on the right. The representative result of three different experiments is reported.

Fig. 4. Construction and characterization of the divalent Ab scFvF7-Fc. A. Vector diagram that expresses the reading frame of the scFvF7-Fc. The positions of the hEF1α / HTLVI promoter, the signal sequences of IL-2 (IL-2 SS) acting as a signal peptide, the multiple cloning site (MCS) and the hinge region are shown. The map of the domains of the resulting protein product is also shown and, on the right, the general structure of the divalent Ab. B.

Western blot analysis with anti-6 × histidine mAb, performed on the supernatants of CHO cells transfected with a vector expressing scFvF7-Fc (lane 1) or transfected with the empty vector (lane 2). Molecular markers (Mk) are shown on the left. The results are representative of four independent assays. C. ELISA performed with supernatants of CHO cells transfected with the vector expressing scFvF7-Fc. The wells of the plate were coated with either recombinant LAG3 or glucose oxidase (GO). The mean values ± SEM calculated from the results of three independent assays are reported.

Fig. 5. Binding properties of the scFvF7-Fc divalent antibody construct. A. Binding curve of purified divalent scFvF7-Fc on recombinant LAG3. One hundred ng of recombinant LAG3 were adhered to the bottom of micro-wells of an ELISA plate and then different amounts of reconstituted Ab F7 were incubated for one hour. The binding with Ab was finally revealed by incubation with an anti-human antibody conjugated with HRP. The mean values ± SEM calculated from quadrupled wells of an experiment representing two independent experiments are shown. O.D: optical density. B. FACS analysis on LAG3 over-expressing 293T cells. Both the parental 293T cells and those engineered for LAG3 expression were incubated with the divalent Ab scFvF7-Fc, and then with a secondary anti-human IgG conjugated with FITC. Data from an experiment representing six independent experiments are shown here. C. 'Dose-response' binding curve of Ab F7 reconstituted on the cell membrane of activated human CD8 T lymphocytes. PBMCs were isolated from peripheral blood of healthy donors, and then the CD8 T fraction was isolated by immuno-magnetic selection. The cells were then activated with PHA and, after six days, incubated with the indicated amounts of divalent scFvF7-Fc. As a control, the activated cells were incubated with secondary Ab only. As an additional control, unstimulated CD8 T cells were incubated with the highest concentration of anti-LAG3 Ab. Representative results from two independent experiments are shown.

Fig. 6. Binding of divalent scFvF7-Fc on antigen-specific CD8 + T lymphocytes. Human CD8 T lymphocytes specific for Nef and Mart1 were stimulated through co-cultivation with HLA-compatible B-LCL previously loaded with specific (stimulated) or mismatched (unstimulated) peptides. After co-cultivation for one night (o.n.), the co-cultures were incubated with reconstituted Ab F7 Ab, followed by incubation with secondary anti-human IgG conjugated with FITC. Then, the cells were incubated with PE-conjugated anti-CD8 mAb. As a control, non-stimulated or PHA activated PBMC-derived CD8 T cells were incubated with secondary or Ab scFvF7-Fc (upper panels). In the panels below, histograms of FITC-linked fluorescence profiles of PE-positive cells are shown. Representative results of four independent experiments are reported.

Fig. 7. Treatment with bivalent Ab scFvF7-Fc increases the activation of antigen-specific CD8 T lymphocytes stimulated with the relative peptide. A. Both Nef and Mart1 specific CD8 T cells were co-cultured with HLA-compatible B-LCL previously treated with the appropriate peptides and in the presence of one of the following antibodies, divalent Ab scFvF7-Fc or mAb anti - Murine LAG3 17B4. As a control, both B-LCL (untreated or treated with appropriate peptides) and CD8 T cells were grown alone or in co-culture. After o.n. incubation, the supernatants were collected, clarified and tested for IFN-γ content. The mean values ± SEM calculated from wells in triplicate from a representative test of three independent experiments are shown. B. Dose-response effect of divalent Ab scFvF7-Fc and inhibition by recombinant LAG3. Co-cultures including B-LCL and Nef specific CD8 T lymphocytes were treated as reported for panel A, however this time in the presence of different amounts of reconstituted Ab F7. Conditions with the highest concentration of divalent scFvF7 and increasing doses of recombinant LAG3 were also included. After o.n. cultivation, the supernatants were tested for IFN-γ content. The mean values ± SEM calculated from wells in triplicate from a representative test of two independent experiments are shown. C. Effects of divalent scFvF7-Fc as detected by ELISPOT test for IFN-γ. IFN-γ specific CD8 T cell response detected by the ELISPOT assay after overnight co-cultures of Nef specific CD8 T cells with B-LCL pre-incubated or not with the specific peptide. Co-cultures were set up in the presence or absence of both divalent Ab scFvF7-Fc and murine anti-LAG3 17B4 mAb. The first co-cultures were also performed in the presence of recombinant LAG3 or the same amount of an unrelated recombinant protein (eg HIV-1 gp120). The results are expressed in spot forming units (SFU) / 105 CD8 T cells, and are representative of two experiments performed with triplicate wells.

EXAMPLE 1: Isolation of the human anti-LAG3 Ab scFv of the present invention by phage display technology, engineering of the divalent Ab scFvF7-Fc according to the present invention and study of the ability of Fc reconstituted F7 to bind antigen-specific CD8 T lymphocytes activated by peptide and to increase IFNγ production in antigen-specific CD8 T lymphocytes.

METHODS

Ab phage library

The IORISS1 'naive' human phage antibody library [39] consists of a large array (ie more than 10 <9> combinations) of Ab polypeptides in the form of scFv expressed on the surface of M13 phages. It was constructed by amplifying light and heavy antibody chains from a group of healthy donors, then randomly joined by means of an appropriate peptide linker and cloning the resulting scFvs into a phagemid vector as previously described [39].

Biopanning strategy for the selection of specific scFvs for LAG3.

The biopanning procedure was performed in ISS in compliance with the obligations relating to GMOs, derived from national and EU regulations.

The IORISS library is derived from a scientific research collaboration between the Istituto Superiore di Sanità (ISS) and IOR; all the experimental procedure relating to the construction of the IORISS library performed in ISS was performed in compliance with the obligations on GMOs, derived from the national authorities and the EU Regulations.

Specific Abs in scFv format were isolated from the phage library as previously reported [42]. Briefly, an aliquot of IORISS-derived phages containing approximately 10 <13> colony forming units (cfu) were contacted with immunotubes (Nunc Maxisorp) previously coated with purified recombinant LAG3 protein (Acrobiosystem), incubated overnight. whole at room temperature (r.t.) at a concentration of 5 µg / mL in 4 ml of a phosphate buffered saline solution (PBS).

After biopanning, phages - derived from a modified version of the vector pdN332, which is a phagemid derived from pHEN1 (for details, see ref [39] regarding the IORISS1 naive human Ab library) - were eluted in 1 mL of 100 mM trimethylamine and the solution was immediately neutralized by adding 0.5 mL of 1M Tris - HCl pH 7.4.

The term phage refers to "phage-like" particles produced with the assistance of a commercial M13 K07 "helper phage" (commercially available, for example from New England Biolabs).

The eluted phages were used to infect TG1 E. coli bacteria [from Lucigen, genotype: [F 'traD36 proAB lacIqZ ΔM15] supE thi-1 Δ (lac-proAB) Δ (mcrB-hsdSM) 5 (rK- mK -) ] in log phase and amplified for a further selection cycle as previously described [42]. Briefly, 50 mL of 2 × YT of medium (Sigma) containing 100 µg / mL of ampicillin (2 × YTA) and 2% glucose were inoculated with sufficient bacterial suspension to produce an optical density (O.D.) at 600 nm of 0 , 05-0.1. The culture was grown to 0.4-0.5 O.D. and then infected with commercial helper phage M13 K07 (marketed by Creative Biolabs, Thermo Fisher Scientific, New England Biolab, Stratagene) in a multiplicity of 20 infections. The recovered phages were concentrated by precipitation with polyethylene glycol (PEG) 6000, and used to subsequent rounds of panning. By plating on agar TG1 cells infected with the phage pool expressing the Abs recovered in the last selection cycle it was possible to grow the single clones containing phagemids. Several bacterial clones were tested for their ability to secrete functional anti-LAG3 scFvs.

For the preparation of soluble scFv, single colonies were cultured in flat bottom wells of a 96 plate (Nunc) for 2 hours at 37 ° C in 180 µL of 2 × YTA medium and 0.1% glucose, then induced with 50 µL of 2 × YTA medium supplemented with 6 mM of isopropyl β dithiogalactopyranoside (IPTG, Sigma). The following day, the plates were centrifuged at 1,800 × g for 10 minutes, and the supernatants were recovered and tested for the presence of scFv.

DNA characterization and sequences

Plasmid DNA from individual bacterial colonies was amplified and analyzed by digestion with Nco I and Not I restriction enzymes (i.e. the restriction sites used to clone antibody fragments in the IORISS1 library) to verify the presence of the scFv encoding inserts. Subsequently, the ORFs of the scFvs of the positive clones were sequenced with an automated DNA sequencer (Eurofins Genomics) using the following primers: scFv forward: 5′-atgaaacaaagcactattgcact-3 ′ (SEQ ID NO: 35); scFv reverse: 5′-ttgatattcacaaacgaatgg-3 ′ (ID SEQ NO: 36). The sequences were finally analyzed using the IMGT Database.

Table 1 shows the IgG gene families that have been identified by IMGT.

Table 1

VH gene families (IMGT)

V-GENE and allele Homsap IGHV3-23 * 04 F J-GENE and allele Homsap IGHJ3 * 02 F

D-GENE and allele

by IMGT / Junction Analysis Homsap IGHD4-23 * 01 ORF VH gene families (IMGT)

V-GENE and allele Homsap IGKV1-9 * 01

J-GENE and allele Homsap IGKJ3 * 01 F

ELISA on recombinant LAG3 protein

The 96-well ELISA plates were coated with 50 µL / well of 10 µg / mL recombinant LAG3 in PBS at 4 ºC. The next day, a blocking solution consisting of 2% milk in PBS (MPBS) was added and after 2 hours the plates were washed with PBS containing 0.1% Tween 20 (TPBS). The plates were then incubated for 2 hours at room temperature with 50 µl of supernatants containing soluble Abs scFv, Ab anti-flag M2 (2.5 µg / mL, Sigma) and HRP conjugated anti-mouse Ab (5 μg / mL , Dako). All antibodies were resuspended in 2% MPBS. The reaction was developed with 3.3 "-5.5" tetramethylbenzidine (BM blue, POD substrate, Roche), and stopped by adding 50 µL of 1 M hydrogen sulphide. The reaction was detected by an ELISA reader (microplate reader , BioRad, model 680) and the results were expressed in terms of absorbance (A) = A450 nm-A620 nm.

For the binding analysis of anti-LAG3 scFv reconstituted with Fc, the coating of the plates was performed with 50 ng of recombinant LAG3 / well. The reconstituted Ab was then incubated for one hour at r.t., followed by incubation with an anti-human Ab conjugated with HRP (Thermo Fisher Scientific). The remaining steps were performed as described above.

Soluble scFv purification

For the production of soluble scFv, TG1 E. coli cells infected with the specific phages were cultured at 37 ºC in 2 × TY containing 100 µg / mL of ampicillin and 0.1% glucose up to an O.D. of 0.5. After induction of the Ab by adding IPTG ≥1 mM, the cells were incubated overnight at 30ºC. Then, the bacterial cultures were centrifuged and the supernatants containing the scFvs recovered. The scFv antibodies were precipitated with ammonium sulfate and then dialyzed in PBS. Abs scFv having a 6 histidine tag were purified by metal affinity chromatography using an agarose resin with Ni2 + nitriloacetic acid (Qiagen). The ScFv fragments were eluted with 250 mM imidazole in PBS, dialyzed, aliquoted and stored at -80 ºC.

Eukaryotic cell cultures

PBMCs were obtained by density gradient centrifugation in Ficoll - Hypaque from heparinized blood samples from healthy donors. The PBMCs were purified from buffy coats obtained from the Transfusion Center of the University of Rome "La Sapienza" as waste material deriving from the plasma / platelet / red blood cell isolation procedures from whole blood of healthy donors, which had given the written informed consent. They were volunteers. In line with the Italian law on the subject (Legislative Decree of the Italian Ministry of Health of 25 January 2001, published in the Official Journal of 3 April 2001) the age of donors was between 21 and 60 years for women and 21-65 years For the men. There were no restrictions for blood donors on gender and ethnicity, with the exception of children, individuals with mental disabilities and pregnant women. All donors were healthy, non-medicated adults, and were preselected for exposure to infectious agents. The blood was obtained by puncture on a vein according to standard medical procedures using a sterile technique and banded compression in the sampling area. A record of the donor and the hematocrit result is filed at the transfusion center. Researchers do not have access to personal data or other information concerning donors in accordance with Italian law (Number 675 of 31 December 1996).

Purified T cells were obtained from PBMC by negative selection using a human T cell selection kit (Miltenyi) according to the manufacturer's protocol, and were cultured in RPMI medium complemented with 10% fetal calf serum inactivated at heat (FCS) (HyClone). Both CHO-K1 (ATCC® CCL-61 ™) and HEK293T (HEK293-EBNA, Invitrogen, Catalog # R620-07) (ATCC) cells were cultured in DMEM enriched with 10% FCS. Both HLA-A02 and HLA-B7 B-LCL cells were cultured in RPMI medium supplemented with 10% FCS. B-LCLs were generated by in vitro infection with EBV of ex vivo cultures of B lymphocytes [43]. The isolation and expansion of clones of CD8 T cells specific for Mart (melanoma-associated recognized by T cells) -1 and for HIV-1 Nef have been previously described [43, 44]. Mart1-specific CD8 T cells recognize the 'restricted' AAGIGILTV 27-35 peptide sequence to HLA-A.02 (SEQ ID NO: 37) (HLA-A.02 peptide sequence from amino acid 27 to 35), while cells Nef-specific CD8 T recognize the 'restricted' TPGPGVRYPL 128-137 peptide for HLA-B7- (SEQ ID NO: 38) (Sequence of HLA-B7 peptides from amino acid 128 to 137). Both antigen-specific CD8 T cell clones were cultured in RPMI plus 10% human AB serum (Gibco) and regularly monitored for their specificity.

Flow cytometric assay on human T cells CD4 T cells isolated from PBMC (for origin, see above) were seeded at a concentration of 2 × 10 <6> / mL and activated with 5 µg / mL of PHA. During the screening phase, scFvF7 was preliminarily tested on TCD4 cells at a single concentration (50 µg / mL), only to have a "yes / no" answer regarding the ability to recognize the antigen on the cell surface. Approximately 1 × 10 <6> cells were resuspended in PBS containing the primary scFv antibody and incubated for 1 hour at r.t .. After washing, the cells were resuspended for 30 minutes at 4 ° C in PBS containing 25 μg / mL. of an M2 anti-FLAG mouse antibody (Sigma), and then incubated for an additional 30 minutes at 4 ° C with 6 μg / mL of FITC-conjugated goat anti-mouse IgG antibodies (Pierce, Illinois, USA). An irrelevant antibody directed against an irrelevant antigen (glucose oxidase, GO) was used as a negative control. After labeling, the cell samples were washed, held at 4 ° C and immediately analyzed at the FACScan (Becton-Dickinson, NJ, USA) equipped with a 15 mW, 488-nm argon laser. Fluorescence compensation was performed using samples incubated with antiglucose scFv and FITC conjugated goat anti-mouse secondary antibody.

As regards flow cytometric analysis on HEK293 T cells (both the parental LAG3-negative cell line and the one derived from it expressing LAG3, kindly provided by the research team of Prof. Pantaleo, Lausanne), 2.5 × 10 <5 > cells were incubated with Fc reconstituted Ab scFvF7 (10 µg / mL) for 1 hour at r.t., and then after washing with a goat anti-human IgG1 FITC- conjugated antibody (Pierce) for 30 minutes at 4 ° C.

Similarly, both PBMC-derived CD8 T cells and antigen-specific CD8 T cells (1x10 <6> cells / test) were incubated with Fc-reconstituted anti-LAG3 scFv, at different concentrations, as specified in each. experimental test (between 0.1 and 20 µg / mL) for 1 hour at r.t.; murine anti-LAG3 mAb 17B4 (EnzoLab, 10 µg / mL) was used as a control. Then, cells were washed and incubated with FITC conjugated anti-human IgG1 secondary (to detect reconstituted scFvF7) or mouse anti-IgG1 goat secondary (for mouse detection 17B4 mAb) (Pierce). In all cases, the cells were then analyzed on the FACscan platform (BD Biosciences).

As for the experiments involving PBMC-derived CD8 T cells, they were performed at the CHUV in Lousanne; this study was approved by the Institutional Review Board of the Centro Hospitalier Universitaire Vaudois and all individuals gave written informed consent. Blood mononuclear cells used in in vitro functional tests were obtained by leukopheresis performed on healthy donors. Blood mononuclear cells were isolated as previously described (Perreau and Kremer, 2005) and cryopreserved in liquid nitrogen.

Western blotting

The supernatants of the transfected cells (50 µL) were separated in 10% SDS-PAGE, then transferred to a nitrocellulose membrane. After the 'blocking' phase with 5% milk, the membranes were washed and incubated for 1 hour at r.t. with HRP-conjugated goat anti-human IgG antibodies (Dilution 1: 2.000, DAKO) and developed using a chemiluminescence reagent (NEN Life Science products). For the analysis of the binding of the anti-LAG3 scFv to the recombinant LAG3 protein in the Western Blot assay, 0.5 μg of recombinant LAG3 and, as a control, of glucose oxidase (GO) (Sigma-Aldrich), were loaded into multiple wells and a 12% SDS-PAGE separation was performed. After transfer to nitrocellulose, different filter strips were incubated with anti-6 × His antibody (exploiting the presence of a 6 × His tag at the C-terminal end of the recombinant LAG3 protein), or with scFvGO (i.e. a human Ab a single chain against the enzyme glucose oxidase) [45], either with scFvF7 or, as a control, with anti-Flag mouse antibodies and HRP-conjugated anti-mouse antibodies. As secondary Abs, HRP-conjugated goat anti-mouse IgG (Dako) or anti-human IgG (Thermo Fisher Scientific) antibodies were used.

Genetic construction and production of the bivalent anti-LAG3 Ab scFvF7-Fc

The procedure followed complied with the obligations relating to GMOs in accordance with national and Community regulations.

The anti-LAG3 scFv coding sequence was amplified by the phagemid vector IORISS1 [39] by PCR with appropriate primers. The amplified fragment was then digested with the enzymes Xho I and Eco RI, purified in a gel and combined with the vector pFUSEss-CHIg-HG1 (Invivogen) - which expresses both human Fc sequences and the CH2 domain, including LALA mutations [46 ] and of the double-digested CH3 domain with Xho I / Eco RI. The resulting construct was transfected into CHO-K1 cells (ATCC® CCL-61 ™) for a pilot scale production of the bivalent Ab scFvF7-Fc. Briefly, 10 <6> cells were transfected with 1 µg of plasmid DNA in a 6-well tissue culture plate with Lipofectamine Plus reagent (Life Technologies) according to the manufacturer's instructions. Supernatants were collected 48 hours later to evaluate Ab production by Western Blot and ELISA analysis.

Large-scale production of the bivalent Ab scFvF7-Fc.

The procedure, carried out at the CHUV laboratories in Lausanne, complied with the regulations relating to GMOs.

The divalent Ab scFvF7-Fc was produced through transient transfection of CHO DG44 cells (ATCC® CRL-9096 ™) followed by incubation for 6 days in serum-free ProCHO5 medium (Lonza). The Ab scFvF7-Fc was purified from the cell medium using a protein A column (Thermo Fisher), eluted with 100 mM pH 3.0 glycine buffer then neutralized with 1M Tris-HCl at pH 8.0. The Abs were then dialyzed twice against PBS, concentrated using a 3 kDa cut-off JumboSep centrifuge filter (Pall Laboratories) and sterile filtered using Millex GP 0.22 μm pore size (Millipore). Before evaluating scFvF7-Fc in functional in vitro tests, the Abs were tested with the FDA-cleared Charles River Laboratory (Endosafe-PTS) Kit, which showed an endotoxin content <5 EU per mg of protein.

ELISA and ELISPOT assays for IFN-γ

CD8 T cells were pre-incubated with different concentrations of reconstituted anti-LAG3 scFv for 2 hours and then co-cultured in a 2: 1 ratio with HLA-compatible B-LCL previously 'pulsed' with 100 ng / mL of appropriate peptide. The co-cultures were incubated o.n., and finally the supernatants clarified and analyzed for IFN-γ content by ELISA (Immunological Sciences). For ELISPOT assays, co-cultures were maintained for 16 hours in ELISPOT micro-wells previously coated with a mAb directed against human IFN-γ (clone D1K, Mabtech). Subsequently, the cells were removed and a biotinylated Ab directed against human IFN-γ was added, followed by the addition of an alkaline phosphatase-conjugated streptavidin. The plate was then developed using the BCIP / NBT (Sigma) substrate. Spot-forming cells were analyzed and counted using an ELISPOT reader (Amplimedical Bioline A-EL-VIS GmbH).

Statistic analysis

Statistical analyzes were performed using GraphPad Prism Software version 5.01. Data are expressed as mean ± standard error of the mean (SEM). The t-student test was used to determine significance. A p value of 0.05 was considered to be statistically significant.

Abbreviations:

B-LCL: B-lymphoblastoid cell lines; DC: dendritic cells; ELISA: immunosorbent enzyme assay; ELISPOT: immunospot enzyme assay; Fc: crystallizable fragment; FCS: fetal calf serum; GO: glucose oxidase; kDa: kilodaltons; HLA: human leukocyte antigen; ICB: immune checkpoint blocker; IFN: interferon; Ig: immunoglobulin; IPTG: isopropyl βdithiogalactopyranoside; IR: inhibitory receptor; LAG3: lymphocyte activation gene 3; mAb: monoclonal antibody; O.D .: optical density; PBMC: peripheral blood mononuclear cells; o.n .: whole night; PBS: buffered saline phosphate solution; PHA: phytohemagglutinin; r.t .: room temperature; scFv: single-chain variable fragment; SE: standard error; TIL: tumor infiltrating lymphocytes.

RESULTS

Isolation of an anti-LAG3 human scFv antibody by phage display technology.

Human recombinant ScFv against LAG3 were obtained by an in vitro affinity enrichment process (bio-panning) as previously described [39], and represented in Fig. 1A. Basically, an aliquot of phages expressing scFvs, adequate to represent the complexity of the IORISS1 library [39], was subjected to binding cycles with the recombinant LAG3 protein used as bait, immobilized on the surface of immunotubes, which is followed by an elution process. A progressive increase in phage titre was recorded after each selection cycle (Table 1). Five rounds of selection were required to isolate an anti-LAG3 phage population detectable in ELISA. Soluble scFvs derived from single bacterial colonies were obtained after plating of TG1-infected bacteria with selected phages. Many scFvs have been shown to bind the recombinant LAG3 protein (Fig. 1B). The DNA encoding the scFvs was isolated from the positive clones and sequencing revealed a common genetic identity (Fig. 2). A representative clone, referred to as scFvF7, was produced in bacteria (TG1) and purified by metal affinity chromatography using the 6 × histidine tag located at its C-terminus. SDS-PAGE analysis showed a single 31 kilodalton (kDa) band, of the expected size for an Ab fragment, in the absence of contaminants and degraded products (Fig. 3A).

The purified scFv was then tested for binding with the recombinant LAG3 antigen in ELISA. The binding assay shows a specific dose-dependent association with LAG3 and lack of binding with the glucose oxidase (GO) protein used as irrelevant antigen (Fig. 3B).

As regards the binding activity of scFvF7 on eukaryotic cells expressing LAG3, the flow cytometric analysis on CD4 T lymphocytes carried out after 3 days of stimulation with phytohaemagglutinin (PHA) has shown that scFvF7 is able to bind also LAG3 in native conformation with a profile comparable to that of a commercial mouse anti-LAG3 Ab used as a control (Fig. 3C). In contrast, scFvF7 does not react with its molecular target in the Western Blot SDS-PAGE assay (Fig.3D), suggesting the recognition of a conformational epitope.

Construction of the bivalent Ab scFvF7-Fc

The use of single domain Abs (scFv) overcomes some of the typical limitations of tetrameric antibodies, for example non-specific uptake, limited access to tissues of interest. On the other hand, scFvs have been shown to have low in vivo stability as a consequence of their rapid elimination from the blood. Therefore, to increase the known instability in vivo for possible clinical use, scFvF7 was fused with the CH2 and CH3 domains of a human Fc. We generated a divalent scFvF7-Fc construct resulting in a recombinant Ab incorporating two scFvF7 molecules, each of which joined the constant CH2 and CH3 immunoglobulin domains. Similarly to the classic antibody, their spontaneous dimerization results in two anti-LAG3 binding sites on the same molecule, with the important advantage that the divalent molecule can be expressed by a single reading frame (Fig. 4A). The presence of a hinge region between scFv and the constant domains ensures flexibility similar to that of natural immunoglobulins. Furthermore, LALA mutations within the CH2 constant regions [46] are particularly useful for possible use in vivo as they reduce the risk of Fc-dependent undesirable effects, such as cell-dependent antibody cytotoxicity. The open reading frame of scFvF7 was cloned 'in frame' with CH2 and CH3 sequences of the Fc region in the context of the pFUSEss-CHIghGI vector which also expresses the secretory leader peptide sequence for human IgG1 (Fig. 4A).

The resulting molecular construct was transfected into CHO cells in order to verify the correct synthesis and secretion of the construct in a eukaryotic system. The Western Blot test performed under non-reducing conditions with the supernatants of the transfected cells, using anti-human Fc antibodies as detector, revealed a dominant signal in a position compatible with the expected molecular weight (∼140 kDa) of the anti-LAG3 Ab. reconstituted (Fig. 4B). These supernatants reacted effectively and specifically in ELISA on the LAG3 protein (Fig. 4C).

Subsequently, the Ab F7 reconstituted with Fc was produced on a large scale by transient transfection in CHO cells, it was then purified and formulated in sterile PBS. The purified divalent Ab construct retains its specificity as evidenced both by its reactivity in ELISA against recombinant LAG-3 (Fig. 5A) and by its binding to human HEK 293 cells stably expressing LAG3 (Fig. 5B). Finally, FACS tests showed that reconstituted Ab F7 specifically binds human CD8 T cells activated with PHA, in a concentration-dependent mode (Fig. 5C).

Ab F7 reconstituted with Fc binds antigen-specific activated CD8 T lymphocytes with its peptide In addition to CTLA-4 and PD1, LAG3 is now considered an important target for mAb-based cancer immunotherapy. ICBs are selected on the basis of their ability to reverse lymphocyte-dependent immune inefficiency, a typical dysfunctional condition that affects TILs infiltrated in solid tumors, in particular the CD8 T subpopulation (12). In this context, the possibility that Ab F7 could be proposed as a new ICB candidate for the new combination anticancer therapies is based on the evaluation of its activity on antigen-specific CD8 T lymphocytes. For this purpose, we performed a functional test on a pair of antigen-specific CD8 T cell clones, i.e. a 'restricted HLA-B7' clone specific for HIV-1 Nef and a restricted HLA-A.02 specific for antigen. of human melanoma Mart-1.

First, we were interested in evaluating the detection capability of LAG3 expression with reconstituted Ab F7 on CD8 T cell clones following specific activation.

The two clones were co-cultured together with compatible B lymphoblastoid (B-LCL) - HLA cell lines previously treated with 100 ng / mL of each specific peptide. As a positive control, CD8 T cells isolated from healthy donor peripheral blood (PBMC) were treated with PHA for three days. Stimulated cells were incubated with reconstituted F7 and labeled with PE-conjugated human IgG secondary antibody. As shown in Fig. 6, after activation, the reconstituted Ab F7 bound both T CD8 clones, a result that was consistent with the expectation of the up-regulation of LAG3. These data validated the two CD8 T cell clones as reliable candidates for functional assays dedicated to evaluating the activity of the bivalent scFvF7 construct.

Increase in IFN-γ production in CD8 + antigen-specific T lymphocytes treated with Ab scFvF7 reconstituted with Fc

B-LCL cells were treated with specific peptides for 3 hours. The peptide-loaded APCs were then co-cultured with the appropriate CD8 T clones in a 1: 1 ratio in the presence or absence of a non-specific control IgG antibody or Abs anti-LAG3 (or scFvF7 reconstituted with Fc or a mouse mAb) in a total volume of 100 µL. After an overnight incubation (o.n.), supernatants were collected and IFN-γ production was evaluated by ELISA. The results reported in Fig. 7A clearly demonstrate that treatment with Fc-reconstituted scFvF7 increases the release of IFN-γ even more efficiently than the mouse anti-LAG3 mAb validated for functional activity. The effect is more pronounced in Nef-specific CD8 T cells, which is consistent with the apparently higher level of LAG3 detected by divalent scFvF7 compared to that observed on the Mart-1 specific CD8 + T cell clone (Fig. 6). .

The specificity of the functional effect of scFvF7-Fc is supported by the data presented in Fig. 7B, where divalent Ab was incubated with cells in the presence or absence of increasing amounts of recombinant LAG3 protein. Ab F7 induces the production of IFN-γ in a concentration-dependent manner which is effectively inhibited with the addition of the soluble LAG3 antigen. The specific effect of divalent Ab F7 on the activation of CD8 T cells was further confirmed by ELISPOT assays for IFN-γ (Fig. 7C). Taken together, the functional data indicate that the divalent human Ab scFvF7-Fc efficiently binds LAG3 on the surface of antigen-specific CD8 T lymphocytes, thus powerfully increasing their activation state. Based on its ability to counteract the inhibitory effect mediated by LAG3 on antigen-activated CD8 T lymphocytes, scFvF7-Fc can be proposed as a new candidate for the treatment of diseases characterized by a lymphocyte dysfunction in which LAG3 is involved.

The combination of the results that were obtained in Western Blot and ELISA are compatible with a binding of scFvF7 with a conformational epitope of LAG3. Furthermore, functional tests performed with antigen-specific CD8 T lymphocytes demonstrated that divalent Fc-fused scFvF7 has biological activity comparable to that of mouse anti-LAG3 17B4 mAb. Considering the human origin of scFvF7, these results are particularly relevant in view of the possible in vivo uses of this antibody and the constructs derived from it.

The biological activity of scFvF7 was evaluated by means of functional tests on CD8 T lymphocytes specific for viral or tumor antigens. Interest has been focused on CD8 T cells to evaluate the efficacy of scFvF7 as a new ICB candidate because the guided immuno-reactivation of CD8 + T cells is expected to have a significant therapeutic effect to counteract the dysfunctional condition of this lymphocyte subpopulation in cancer but even in chronic infectious diseases.

CONCLUSIONS

It is expected that an increasing number of Abs against emerging regulatory molecules may play a key role both in promoting systematic analyzes for the definition of robust bio-markers useful for predicting the therapeutic efficacy and / or toxicity of treatments, and for the preparation of combined therapies, tailor-made for patients, in the context of precision medicine. In the present study the isolation and functional activity of a new recombinant anti-LAG3 Ab is described. While further characterization studies are needed to confirm the potential of scFvF7 as a clinical candidate for the treatment of some pathological conditions in combination with other ICBs, the data presented supports this agent as a valid scaffold for the development of novel LAG3-based interventions.

Finally, the flexibility of the biotechnological platform described allows easy exploration of alternative therapeutic constructs, including bispecific or bifunctional Abs. These new Abs could further enhance functional activity by replacing an antigen-binding portion with another ligand / cytokine and promoting the coupling of the related heterodimers by inserting specific mutations; for example a bispecific antibody bearing on one antibody arm the binding site of scFvF7 (specific for Lag3) and on the other a specific binding site for another iCP (eg PD1); or an antibody construct bearing on one arm the binding site of scFvF7 (specific for Lag3) and on the other an immunostimulating cytokine such as IL-15 (eg by Knob / Hole mutations [47] on constant regions).

Claims (1)

CLAIMS 1) Human recombinant antibody sequence directed against a conformational epitope of LAG3 comprising or consisting of a VH sequence comprising the CDR sequence AKDQDGGKKTDAFDI (SEQ ID NO: 3), said VH sequence optionally also comprising the following CDR sequences: ISAGGTGT (SEQ ID NO: 2), or ISAGGTGT (SEQ ID NO: 2) and GFTFSSYA (SEQ ID NO: 1), or GFTFSSYA (SEQ ID NO: 1) and a VL sequence comprising the CDR sequence QTGYYPLT (SEQ ID NO: 5), said VL sequence optionally further comprising the following CDR sequences: AAS, or AAS and QGINYY (SEQ ID NO: 4), or QGINYY (SEQ ID NO: 4), in which said VH and VL sequences are optionally connected by a linker. 2)Sequenza di anticorpo secondo la rivendicazione 1, in cui detta sequenza VH comprende o consiste in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSS (SEQ ID NO: 6) e detta sequenza VL comprende o consiste in DIVMTQSPSLLSASVGDRVTITCRASQGINYYLAWYQQKPGKAPKLLIYAASTLQ SGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQTGYYPLTFGPGTKVDI (SEQ ID NO: 7), detta sequenza VL optionally further comprising a restriction site at the C terminal end, such as KAAA (SEQ ID NO: 8). 3) Antibody sequence according to any one of claims 1-2, wherein said linker is GGGGSGGGGSGGGGS (SEQ IDNO: 9). 4)Sequenza di anticorpo secondo una qualsiasi delle rivendicazioni 1-3, detta sequenza di anticorpo comprendendo o essendo consistente in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMQSPSLLSASVGDRVTITCRASQ GINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDI (SEQ ID NO: 10), facoltativamente comprendendo inoltre un sito di restrizione all'estremità C terminale, come KAAA (SEQ ID NO: 8). 5)Sequenza di anticorpo secondo una qualsiasi delle rivendicazioni 1-4, detta sequenza di anticorpo comprendendo o essendo consistente in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAADDDSDDDYKDDDDQHHHHHH (SEQ ID NO: 11). 6) Antibody sequence according to any one of claims 1-5, which further comprises human domains constant of immunoglobulins, such as CH2-CH3 or CH3 in which optionally the CH2 domain comprises LALA mutations, in which said domains are linked to the VL sequence by a zipper, such as EPKSCDKTHTCPPCP (SEQ ID NO: 15). 7)Sequenza di anticorpo secondo la rivendicazione 6, detta sequenza di anticorpo comprendendo o essendo consistente in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAAASEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (ID SEQ NO: 16). 8)Sequenza di anticorpo secondo una qualsiasi delle rivendicazioni 6-7, detta sequenza di anticorpo comprendendo o essendo consistente in MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISA GGTGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQDGGKKTDA FDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSLLSASVGDRVTITCRAS QGINYYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQTGYYPLTFGPGTKVDIKAAAASEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:17) 9)Sequenza di anticorpo secondo una qualsiasi delle rivendicazioni 6 -8, which is in bivalent form. 10) Nucleotide sequence encoding the antibody sequence as defined in any one of claims 1-9. 11) Nucleotide sequence according to claim 10, wherein the nucleotide sequence encoding VH comprises the nucleotide sequence CDR GCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCTTTTGATATC (SEQ ID NO: 20), said nucleotide sequence encoding VH, optionally including the following nucleotide sequences SEQAGTGACTGCTAGTIDCGTGACTGACTG NO: 19), or ATTAGTGCTGGTGGTACTGGCACA (SEQ ID NO: 19) and GGATTCACCTTTAGCAGCTATGCC (SEQ ID NO: 18), or GGATTCACCTTTTAGCAGCTATGCC (SEQ ID NO: 18), and the nucleotide sequence CACTGACTTAGTCCCACTT IDCTAGTC NO: 22), said nucleotide sequence encoding VL optionally also comprising the following CDR nucleotide sequences: GCTGCATCC, or GCTGCATCC and CAGGGCATTAACTATTAT (SEQ ID NO: 21), or CAGGGCATTAACTATTAT (SEQ ID NO: 21). 12)Sequenza nucleotidica secondo una qualsiasi delle rivendicazioni 10-11, in cui la sequenza nucleotidica che codifica VH comprende o consiste in: ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (ID SEQ NO: 23) 13)Sequenza nucleotidica secondo una qualsiasi delle rivendicazioni 10 -12, in cui la sequenza nucleotidica codificante VL comprende o consiste in: GATATTGTGATGACGCAGTCTCCATCCCTCCTGTCTGCATCTGTAGGAGACAGAG TCACCATCACTTGCCGGGCCAGTCAGGGCATTAACTATTATTTAGCCTGGTATCA GCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAA AGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCA CAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGACTGG TTATTACCCTCTCACTTTCGGCCCTGGGAC CAAAGTGGATATC (ID SEQ NO: 24), optionally also including a nucleotide sequence that encodes a restriction site at the end of 3 ', for example AAAGCGGCCGCA (SEQ ID NO: 25). 14) Nucleotide sequence according to any one of claims 10-13, wherein the nucleotide sequence encoding the linker connecting VH and VL is: GGTGGAGGTGGATCTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCC (ID SEQ NO: 26). 15)Sequenza nucleotidica secondo ciascuna delle rivendicazioni 10-14, detta sequenza nucleotidica comprendendo o essendo consistente in: ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATC (SEQ ID NO: 27), facoltativamente comprendendo anche una sequenza nucleotidica codificante a restriction site at the 3 'end, such as AAAGCGGCCGCA (SEQ ID NO: 25). 16)Sequenza nucleotidica secondo ciascuna delle rivendicazioni 10-15, detta sequenza nucleotidica comprendendo o essendo consistente in: ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGATGACGATTCTGACGATGACTA CAAGGACGACGACGACCAGCACCATCACCATCACCATTAG (SEQ ID NO: 28) 17)Sequenza nucleotidi antibody according to any one of claims 10 to 16, wherein said antibody nucleotide sequence further comprises a nucleotide sequence encoding human constant immunoglobulin domains, such as CH2-CH3 or CH3 in which optionally the CH2 domain comprises LALA mutations, which are linked to the sequence nucleotidica che codifica VL da un sequenza nucleotidica che codifica una cerniera come GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA (SEQ ID NO:32) 18)Sequenza nucleotidica anticorpale secondo una qualsiasi delle rivendicazioni 10-17, detta sequenza nucleotidica comprendendo o essendo consistente in: ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTC ACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTTTCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGCTAGCGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA (SEQ ID NO:33) 19)Sequenza nucleotidica anticorpale secondo una qualsiasi delle rivendicazioni 10-18, detta sequenza nucleotidica comprendendo o essendo consistente in: ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAA CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGCT GGTGGTACTGGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAGAGCCGAGGA CACGGCCGTATATTACTGTGCGAAAGATCAGGACGGTGGTAAGAAGACTGATGCT TTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGTGGAGGTGGAT CTGGCGGCGGCGGCTCAGGCGGAGGAGGTTCCGATATTGTGATGACGCAGTCTCC ATCCCTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGT CAGGGCATTAACTATTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTA AGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAG CGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAA GATTTTGCAACTTATTACTGTCAGCAGACTGGTTATTACCCTCTCACTT TCGGCC CTGGGACCAAAGTGGATATCAAAGCGGCCGCAGCTAGCGAGCCCAAATCTTGTGA CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCTGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA (SEQ ID NO: 34) 20)Vettore comprendente la sequenza nucleotidica secondo una qualsiasi delle rivendicazioni 10-19. Vector according to claim 20, wherein said vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector or a non viral vector. 22) Cell comprising the vector according to any one of claims 20-21, wherein said cell is preferably a eukaryotic cell, more preferably a CHO cell. 23) Pharmaceutical composition comprising or consisting of an antibody sequence as defined in any one of claims 1-9, a nucleotide sequence as defined in any one of claims 10-19, a vector as defined in any one of claims 20-21 or a cell as defined in claim 22, as active ingredient, together with one or more excipients and / or adjuvants. 24) Pharmaceutical composition according to claim 23, which further comprises at least one other therapeutic active principle such as antibodies, cytokines, chemical drugs, preferably immune checkpoint blockers, such as anti-CTLA4, anti-PD1 antibodies. 25) Antibody sequence as defined in any one of claims 1-9, a nucleotide sequence as defined in each of claims 10-19, a vector as defined in each of claims 20-21, a cell as defined in claim 22 or a composition pharmaceutical as defined in each of claims 23-24, for use as a medicament. 26) Antibody sequence as defined in any one of claims 1-9, a nucleotide sequence as defined in any one of claims 10-19, a vector as defined in any one of claims 20-21, a cell as defined in claim 22 or a pharmaceutical composition as defined in any one of claims 23-24, for use in the treatment of a disease which is characterized by a lymphocytic dysfunction in which LAG3 is involved. 27) Antibody sequence as defined in any one of claims 1-9, a nucleotide sequence as defined in any of claims 20-21, a cell as defined in claim 22 or a pharmaceutical composition as defined in any of claims 23-24, for use according to claim 26, wherein said disease characterized by lymphocyte dysfunction in which LAG3 is involved is selected from the group consisting of human tumors such as NSCLC, colorectal carcinoma, breast cancer, hepatocellular carcinoma, head and neck carcinoma squamous cell, hematological and solid cancers such as melanoma, follicular lymphoma, infectious diseases such as chronic infectious diseases, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). 28) Combination of an antibody sequence as defined in any one of claims 1-9, a nucleotide sequence as defined in any one of claims 10-19, a vector as defined in any one of claims 20-21, a cell as defined in claim 22 or a pharmaceutical composition as defined in any one of claims 23-24, with at least one other therapeutic active ingredient such as antibodies, cytokines, chemical drugs, preferably immune checkpoint blockers, such as anti-CTLA4, anti-PD1 antibodies, for the 'sequential or separate use in the treatment of a disease characterized by a lymphocyte dysfunction in which LAG3 is involved. 29) Combination, according to claim 28, for use according to claim 28, wherein said disease characterized by lymphocyte dysfunction in which LAG3 is involved is selected from the group consisting of human tumors such as NSCLC, colorectal cancer, breast cancer , hepatocellular carcinoma, squamous cell cancer of the head and neck, hematological and solid tumors, such as melanoma, follicular lymphoma, infectious diseases such as chronic infectious diseases, such as human immunodeficiency virus (HIV) infection, human immunodeficiency virus (HIV) hepatitis B (HBV), and hepatitis C virus (HCV). 30) Use of the antibody sequence according to any one of claims 1-9 for the in vitro diagnosis of a disease which is characterized by a lymphocytic malfunction in which LAG3 is involved. 31) Use according to claim 29, wherein said disease characterized by a lymphocyte malfunction in which LAG3 is involved is selected from the group consisting of human tumors such as NSCLC, colorectal carcinoma, breast cancer, hepatocellular carcinoma, squamous cell tumor of the head and neck, hematological and solid cancers, such as melanoma, follicular lymphoma, infectious diseases such as chronic infectious diseases, such as human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV), and virus of hepatitis C (HCV). 32) Kit for in vitro diagnosis of a disease which is characterized by the exhaustion of lymphocytes driven by LAG3, said kit comprising a sequence of antibodies according to any one of claims 1-9.33) Kit according to claim 32, in which said disease characterized a lymphocyte malfunction in which LAG3 is involved is selected from the group consisting of human cancers such as NSCLC, colorectal cancer, breast cancer, hepatocellular carcinoma, squamous cell cancer of the head and neck, hematological and solid cancers, such as melanoma, follicular lymphoma, infectious diseases such as chronic infectious diseases, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infection.