TW201305211A - Novel homogeneous humanized antiproliferation antibodies - Google Patents

Abstract

The present invention relates to novel humanized antibodies able to inhibit tumor growth, as well as the amino and nucleic acid sequences coding for such antibodies. From one aspect, the invention relates to novel anti-JAM-A homogeneous humanized antibodies able to inhibit tumor growth. The invention also comprises the use of such antibodies as a drug for the preventive and/or therapeutic treatment of cancers, as well as compositions comprising such antibodies in combination with other anticancer compounds. The invention also relates to a method for the preparation of such novel humanized antibodies.

Description

Novel homologous anthropomorphic anti-proliferative antibody

The present invention relates to novel anthropomorphic antibodies that inhibit tumor growth, as well as amino acid and nucleic acid sequences encoding such antibodies. In one aspect, the invention relates to novel anti-JAM-A homogenous anthropomorphic antibodies that inhibit tumor growth. The invention also encompasses the use of such antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancer, and compositions comprising such antibodies in combination with other anti-cancer compounds. The invention also relates to a method of preparing such novel anthropomorphic antibodies.

One of the most promising treatments against human cancer and other diseases is antibody therapy.

The terms "antibody", "antibodies" or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (eg, full-length or intact monoclonal antibodies), A strain antibody, a multivalent antibody or a multi-specific antibody (for example, a bispecific antibody as long as it exhibits a desired biological activity).

Typically, this molecule consists of a glycoprotein comprising at least 2 heavy (H) chains and 2 light (L) chains interconnected by a disulfide bond. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain conserved region. The heavy chain conservation region consists of three fields, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain conserved region. The light chain conservation area contains a field, CL. The VH and VL regions can be further distinguished as highly variable regions, referred to as complementarity determining regions (CDRs), with a more conserved region interspersed, referred to as the framework region (FR). Each VH and VL line consists of three CDRs and four FRs, arranged in the order of the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy and light chains contains a domain that can bind to one of the antigens. The conserved region of the antibody can mediate the binding of the immunoglobulin to the host tissue or factor, including various cells of the immunological system (such as effector cells) and the first complement (Clq) of the typical complement system.

These may also include certain antibody fragments, as described in more detail herein, which exhibit the desired binding specificity and affinity, regardless of source or immunoglobulin class (ie, IgG, IgE, IgM, IgA, and many more).

In general, the preparation of a single antibody or a functional fragment thereof, especially the origin of the murine, can be referred to the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring). The technique specifically described in Harbor NY, pp. 726, 1988) or the preparation technique from fusion tumors as described by Kohler and Milstein (Nature, 256: 495-497, 1975).

Typically, the anti-system is derived from a non-human source, which is applied to humans to make it necessary to reduce its immunogenicity when administered to a human subject.

Mouse monoclonal antibodies are highly immunogenic in humans, thus limiting their potential use in cancer therapy, especially when repeated administration is required. A variety of methodologies have been developed to deal with this problem. First, to reduce the immunogenicity of mouse monoclonal antibodies, chimeric monoclonal antibodies were generated using the variable Ig domain of mouse monoclonal antibodies fused to the human conserved Ig domain.

Although some chimeric monoclonal antibodies have been shown to be less immunogenic in humans, the mouse variability in the Ig domain can still result in significant human anti-mouse responses.

Full anthropomorphization can be performed by introducing six CDRs from the mouse heavy and light chain variable Ig domains into appropriate framework regions of the human variability Ig domain. This CDR grafting technique utilizes a conserved structure in the variable Ig domain with four framework regions (FR1-FR4) for use as scaffolds for supporting CDR loops, which are the major junctions of the antigen.

However, a disadvantage of the CDR grafting technique is the fact that the amino acid of the framework region can promote antigen binding, and that the amino acid of the CDR loop can affect the association of two variable Ig domains. In order to maintain the affinity of anthropomorphic monoclonal antibodies, CDR grafting techniques rely on appropriate selection of human framework regions and computer modeling of antigen binding sites to assist in the site-directed mutagenesis of a single amino acid (site-directed). Mutagenesis).

The applicant has reported by functional means to produce a murine anti-tumor antibody, called 6F4 or m6F4, which binds to the protein JAM-, as described in the published patent application WO 2008/062063, the entire disclosure of which is hereby incorporated by reference. A and the ability to inhibit proliferation of tumor cells in vitro and/or in vivo in a significant manner.

JAM-A is a membrane protein belonging to the immunoglobulin superfamily (IgSF), which belongs to the family of binding adhesive molecules (JAM). In humans, the JAM family contains several members, including JAM-A, JAM-B, JAM-C, A33, and A34 proteins. Among the JAM family members, JAM-A has the highest homogeneity with JAM-B and JAM-C, with approximately 35% sequence identity for amino acids and 45% similarity to these two proteins. The JAM-A protein is also known as JAM A, F11R, F11 receptor, JAM-1, JAM 1, PAM-1 or CD321.

Identification of two isoforms of JAM-A precursors of different lengths in the extracellular region, each of which is A and B: proteins expressed on the surface of human cells have a single polypeptide chain with an intracellular C-terminal domain, a single transmembrane domain (21 amino acids) and an N-terminal extracellular domain containing two "Ig-like" domains.

JAM-A has an N-glycosylation site, an Asn residue (in position 185 for homozygous A and position 145 for homozygous B), and two disulfide bridges, between Ig N- One of the Cys residues 50 and 109 of the end domain and one of the residues Cys 153 and 212 of the second Ig domain.

The presence of two extracellular Ig domains was confirmed by crystallography (Kostrewa et al., 2001, EMBO J. 16:4391-4398; Prota et al., 2003, Proc. Natl. Acad. Sci. USA, 100: 5366-5371). These two domains are linked by a tripeptide linker (sequence VLV [127-129], homology A). These structural studies have also confirmed that JAM-A is involved in hemophilic interactions on the cell surface involved in the extracellular region; it is produced in recombinant form and is capable of forming homodimers in solution (Bazzoni This region of et al., 2000, J. Biol. Chem. 275: 30970-30976) also makes it possible to identify amino acids involved in such interactions: Arg 59, Glu 61, Lys 63, Leu 72, Tyr 75 Met 110, Glu 114, Tyr 119 and Glu 121. The tripeptide RVE [59-61] is relatively conserved in the JAM family (RLE for JAM-B, RIE for JAM-C) and constitutes the smallest part for the formation of homodimers ( Motif) (Kostrewa et al., 2001, EMBO J. 16:4391-4398).

In terms of epithelial cells and endothelial cells, JAM-A is mainly present in tight junctions (Liu et al., 2000, J. Cell Sci., 113: 2363-2374). The cytoplasmic region contains a type II PDZ domain at the C-terminal position (sequence FLV [298-300], a homologous a, which is responsible for the interaction of JAM-A and various cytosolic proteins associated with tight junctions, Also included is a PDZ domain such as ZO-1, AF-6, MUPP-1 and PAR-3 (Ebnet et al, 2000, J. Biol. Chem., 275: 27979-27988; Itoh et al, 2001, J. Cell Biol., 154: 491-498; Hamazaki et al, 2002, J. Biol. Chem., 277: 455-461). For murine antibodies against regions [111-123] involved in dimer formation, the so-called The J3F.1 and J10.4 antibodies are capable of inhibiting homodimerization of JAM-A and reconstitution of the epithelial barrier (Mandell et al., 2004, J. Biol. Chem., 279: 16254-16262).

The JAM-A line interacts with integrin αVβ3 and involves the migration of endothelial cells to vitronectin, the ligand of integrin αVβ3 (Naik and Naik, 2005, J. Cell Sci. 119: 490-499). The anti-JAM-A antibody J3F.1, in the same manner as an anti-αVβ3 antibody, inhibits endothelial cell migration and in vitro angiogenesis induced by bFGF (Naik et al., 2003, Blood, 102: 2108-2114). ). A variety of signaling pathways are present in endothelial cells: MAP kinase, PI3-kinase and PKC (Naik et Naik, 2005, J. Cell Sci., 119: 490-499; Naik et al, 2003, Blood, 102: 2108-2114; Naik et al., 2003, Arthioscler. Thromb. Vasc. Biol., 23: 2165-2171).

JAM-A is also expressed in mononuclear cells, lymphocytes, neutrophils and platelets (Williams et al., 1999, Mol. Immunol., 36: 1175-1188). However, the JAM-A protein was originally recognized as a receptor for the F11 antibody, which is an antibody capable of activating platelets and inducing aggregation thereof (Naik et al., 1995, Biochem. J., 310: 155-162; Sobocka) Et al., 2000, Blood, 95: 2600-2609). The peptides [28-60] and [97-109] belong to the F11 antibody epitope and are involved in platelet activation and aggregation and involve homodimerization (Babinska et al., 2002, Thromb. Haemost., 87:712-721).

The rat antibody BV11, against the murine form against JAM-A, inhibits the migration of mononuclear spheres across the endothelium in vitro and in vivo (Del Maschio et al., 1999, J. Exp. Med., 190: 1351-1356). ). Ostermann and colleagues (2002, Nature Immunol., 3: 151-158) demonstrate that JAM-A is a ligand for αLβ2 or LFA-1 (lymphocyte function-associated antigen 1) integrin, which is resistant to the development of inflammatory responses. Some chemokines react during the overexpression and are required for hemoglobin exudation or migration to an inflamed site. JAM-A, via the second Ig-like domain, promotes adhesion of T lymphocytes and neutrophils and migration across the endothelium (Ostermann et al., 2002, Nature Immunol., 3: 151-158), and thus in supplementation White blood cells play an important role in the location of inflammation.

JAM-A protein is also involved in viral infection. JAM-A is truly a receptor for the reovirus, a cause of certain types of encephalitis that interacts with the attached protein σ1 (Barton et al., 2001, Cell 104:441- 451). Anti-JAM-A antibody J10.4 inhibits the binding of Ley virus to JAM-A (Forrest et al, 2003, J. Biol. Chem., 278: 48434-48444).

Although the patent application WO 2008/062063 has been disclosed, and must be considered as part of the prior art, the 6F4 antibody can be described as an antibody that is capable of inhibiting the proliferation of tumor cells in vitro and/or in vivo by one of the isolated mice. Or a derivative compound or functional fragment thereof; the antibody, or a derivative compound or functional fragment thereof, comprising i) a light chain comprising, according to IMGT, a sequence having sequence number 1 or after optimal alignment The sequence of sequence identification number 1 has at least 80% identity of one of the CDR-L1 sequences; the sequence with sequence identification number 2 or at least 80% identity with the sequence of sequence identification number 2 after optimal alignment a sequence of CDR-L2; and a sequence having sequence number 3 or a CDR-L3 having at least 80% identity to the sequence of sequence number 3 after optimal alignment, and ii) a heavy a strand comprising, according to IMGT, a sequence having sequence identification number 4 or a sequence of CDR-H1 having at least 80% identity to the sequence of sequence identification number 4 after optimal alignment; having sequence identification number 5 Sequence or CDR-H2 with one sequence of at least 80% identity and sequence with sequence identification number 6 after sequence alignment with sequence identification number 5 or sequence with sequence identification number 6 after optimal alignment There is at least 80% identity of one of the sequences of CDR-H3.

IMGT's unique number has been defined to compare variants, regardless of antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommi , C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In terms of IMGT's unique numbering, conserved amino acids always have the same position, for example, cysteine 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, half Cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides the frame area (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementarity decision area: CDR1-IMGT : 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117, the standardization demarcation. Since the gap indicates an unoccupied position, the length of the CDR-IMGT (shown between the brackets and separated by dots, such as [8.8.13]) becomes decisive information. The unique numbering of IMGT is used in 2D graphical representation, called IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M .-P., Current Bioinformatics, 2, 21-30 (2007)], and in the 3D structure of the IMGT/3D structure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T Cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

An amino acid sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, having certain modifications with respect to a reference sequence, in particular at least one amine It is preferred that the base acid is censored, added or substituted, truncated or extended. In the case of substituting one or more contiguous amino acids or discontinuous amino acids, the substituted amino acid is preferably replaced by an "equal" amino acid. Here, the expression "equal amino acid" is intended to indicate any amino acid which can be substituted for one of the amino acids of the base structure, however, does not substantially modify the biological activity of the corresponding antibody and For example, in the following, especially the biological activity as defined in the examples. Such equal amino acids may be determined by their structural homology to the amino acid to be substituted, or by comparison of biological activities between the various antibodies that are likely to be produced. The result is decided.

According to an "antigen-binding fragment" or "functional fragment" of an antibody, it is intended to specifically indicate an antibody fragment such as Fv, scFv (sc represents a single strand), Fab, F(ab') ₂ , Fab', scFv-Fc fragment. Or dibodies, or any fragment that has been modified by chemical modification to increase half-life, such as the addition of polyalkylene glycols, such as polyethylene glycol ("PEGylated") (PEGylated) Fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG) ("PEG" stands for polyethylene glycol), or by any of the incorporation of liposomes Fragments having at least one of the characteristic CDRs of an antibody of the invention, and, in particular, capable of exerting, in a general manner, even part of the activity of the antibody (which is produced by the antibody), For example, in particular, the ability to identify and combine JAM-A.

A "derivative" or "derived compound" of an antibody specifically means a binding protein comprising at least one of a peptide scaffold and the CDRs of the original antibody to maintain its ability to bind. Derivative compounds well known to those skilled in the art are described in more detail in the following description.

It must be understood herein that the invention is not related to the native form of the antibody, that is, it is not obtained by its natural environment, but is isolated or obtained by purification from a natural source or by a gene. The non-natural amino acids obtained by recombinant or chemical synthesis and thus may carry as described below.

The murine antibody 6F4 can also be described as an antibody comprising a light chain of amino acid sequence number 7 and a heavy chain of amino acid sequence number number 8.

This monoclonal antibody 6F4, or a derivative compound or functional fragment thereof, is secreted by the fusion tumor which was submitted by CNCM on July 6, 2006, under the number I-3646.

According to the conventional CDR graft as explained above, the Applicant has produced an anthropomorphic form of the murine 6F4 Mab. This first anthropomorphic form, referred to hereinafter as Hz6F4-1 (either Hz6F4 1 or 6F4Hz-1 or also 6F4HZ1), has traditionally been introduced into the human reproductive series by introducing the 6 CDRs of murine 6F4. obtain. This anthropomorphic method is described in patent application WO 2008/062063.

Surprisingly, Hz6F4-1 does not exhibit the common shortcomings of the anthropomorphic antibodies as described above (loss of association or modification of the association of two mutable Ig domains) but exhibits its developability and Specific disadvantages of manufacturability.

More specifically, as will be clarified in the examples below, purified Hz6F4-1 has a very unique heterogeneous profile, including many charged variants, which may be in the process of pharmaceutical development and antibody production. a sign of future difficulties.

Another major drawback of Hz6F4-1 is its acidic isoelectric point, which does not allow for efficient viral inactivation, which is forced by an antibody produced within a mammalian cell line. Sexual steps.

In general, the acidic isoelectric point of an IgG-type monoclonal antibody can be considered to be an isoelectric point below 7.

A typical purification platform follows a conventional approach with three major chromatographic steps. Protein A chromatography is used in the initial capture step. The eluate is then kept at a low pH to not activate the virus and then adjusted prior to the cation exchange chromatography step (CEX) to increase the pH. After cation exchange, a diafiltration step is required to prepare the product for the buffing step, which is carried out using a flow-through mode anion exchanger (AEX). The anion exchange step can employ a chromatographic medium or a membrane absorbent. In most cases, this platform is efficient and robust because typically mAbs have high isoelectric points (>7).

Some problems were caused by the purification of Hz6F4-1, which has an isoelectric point below 7 and is close to 5.8. Low pH virus inactivation is virtually impossible with Hz6F4-1 because it is unstable when the pH is below 4.

In order to maintain an acceptable binding capacity to the cation exchange chromatography medium, the loading pH must be below 4, which is a problem because of the instability of Hz6F4-1 at this pH. On the other hand, the lower the CEX loading pH, the higher the impurities retained with the mAb on the chromatographic medium, such as host cell protein (HCP), virus, DNA and endotoxin. Processing HCP clearance is reduced because most Chinese hamster ovary cell proteins (CHOP) have an isoelectric point closer to the isoelectric point of Hz6F4-1. Virus removal by the anion exchange step of the flow mode is likely to have been affected by the required loading pH.

The problems with these techniques were first reported in the field of anthropomorphism of antibodies.

The present invention relies on the observations made by the Applicant and the implementation of modifications to solve this problem.

More particularly, the present invention relates to a method for preparing a mouse variant of 6F4, a novel variant of a humanized antibody designated Hz6F4-2 (or Hz6F4 2 or 6F4Hz-2 or also 6F4HZ2).

According to a general aspect of the present invention, a method for preparing a homologous variant of a parental humanized antibody, or a derivative or antigen-binding fragment thereof, comprising a light The chain variable region has three CDRs sequence identification numbers 1, 2 and 3, and a heavy chain variable region, and the heavy chain variable region has three CDRs sequence identification numbers 4, 5 and 6, and the anthropomorphic antibody is capable of binding JAM-A, wherein the method comprises the following steps:

a) substituting a neutral amino acid residue or a basic amino acid residue for the light chain variable domain of the parentalized humanized antibody or at least one amine of the heavy chain variable region of the parentalized humanized antibody Acid residue;

b) whether the substitution results in an increase in the pI of the variant anthropomorphic antibody compared to the parentalized humanized antibody to select the variant of the anthropomorphic antibody obtained in step a) as its high homozygous variant .

It must be understood that as will become apparent in the following examples, an antibody having the characteristics "high homogeneity" means an antibody which exhibits a homogenous lateral shape in terms of saccharification, quality, and the like, resulting in Improved developability, manufacturability, and overall shape of the drugability. For example, in the present case, the high homogeneity variant of the invention contains fewer multi-charged variant saccharification residues and fewer higher quality variants compared to the parent antibody. This wording is obvious in light of the following examples which will be apparent to those skilled in the art.

The isoelectric point (pI), sometimes abbreviated to IEP, is the pH at which the antibody carries a net charge equal to zero. The theoretical pI can be calculated via software and then determined by an analitycal method such as isoelectric focusing (IEF) or capillary IEF (cIEF).

Likewise, strategies for selecting amino acid substitutions will also be apparent to those skilled in the art upon reading the following examples.

An amino acid substitution that increases the pi of the antibody can comprise substituting an acidic amino acid with a neutral amino acid or a basic amino acid and replacing an acidic amino acid with a basic amino acid.

The neutral amino acid may be selected from the group consisting of alanine (A), proline (V), leucine (L), isoleucine (I), and proline (P). , methionine (M), phenylalanine (F), tryptophan (W), glycine (G), serine (S), threonine (T), cysteine (C) , aspartic acid (N), glutamic acid (Q) or tyrosine (Y).

The acidic amino acid may be selected from the group consisting of aspartic acid (D) or glutamic acid (E).

The basic amino acid may be selected from the group consisting of lysine (K), arginine (R) or histidine (H).

In a preferred embodiment, in step a) of the method according to the invention, at least one amino acid selected from the group consisting of D1, Q3, Q24 and E55 of the light chain variable region having sequence number identification number 9 The residue is substituted with a neutral amino acid residue or a basic amino acid residue.

Accordingly, the present invention also relates to a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, comprising the light chain variability field of the parent antibody of SEQ ID NO: 9, selected from D1 At least one amino acid residue of Q3, Q24 and E55 is substituted with an amino acid residue which causes an increase in the positive charge of the light chain.

It must be understood here that an increase in pI results in an increase in net positive charge. Thus, a substitution that increases pI should be considered equivalent to a substitution that increases positive charge.

In another preferred embodiment, in step a) of the method according to the invention, at least one selected from the group consisting of E1, Q38, G44, E62 and D77 of the heavy chain variable region having sequence number identification number 10. The amino acid residue is substituted with a neutral amino acid residue or a basic amino acid residue.

Accordingly, the present invention also relates to a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, comprising the heavy chain variability field of the parent antibody of SEQ ID NO: 10, selected from E1 At least one amino acid residue of Q38, G44, E62 and D77 is substituted with an amino acid residue which causes an increase in the positive charge of the heavy chain.

In another aspect, the invention comprises a method of preparing a high homogeneity variant of a parental anthropomorphic antibody according to the invention, wherein in step a):

- at least one amino acid residue selected from the group consisting of D1, Q3, Q24 and E55 in the light chain variable region having sequence number identification number 9 is a neutral amino acid residue or a basic amino acid Replacing residues, and

- at least one amino acid residue selected from the group consisting of E1, Q38, G44, E62 and D77 having the sequence of the sequence identification number 10 of the heavy chain variant is a neutral amino acid residue or a basic amine Substituted by a base acid residue.

Another particularly preferred aspect of the invention pertains to a method of preparing a high homogenous variant of a parentalized humanized antibody as described above, wherein in step a):

i) at least one amino acid residue selected from the group consisting of D1, Q3, Q24 and E55 of the light chain variability field having sequence number identification number 9 is each amino acid residues A, R, K and Q Replace;

Ii) at least one amino acid residue selected from the group consisting of E1, Q38, G44, E62 and D77 having the sequence of the heavy chain variant having sequence identification number 10, each having an amino acid residue Q, R, R, Q and S are replaced.

Thus, the invention also features a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, characterized in that it comprises:

i) the sequence of light chain variability of the parent antibody of sequence sequence identification number 9, wherein at least one amino acid residue selected from the group consisting of D1, Q3, Q24 and E55 is each amino acid residue A, R, K and Q to replace; and

Ii) the heavy chain variability field of the parent antibody of sequence sequence identification number 10, wherein at least one amino acid residue selected from the group consisting of E1, Q38, G44, E62 and D77 is each an amino acid residue Q , R, R, Q and S are substituted.

More particularly, a preferred variant of the invention resides in a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, characterized in that it comprises:

i) a light chain variability field comprising sequence sequence identification number 11, and

Ii) A heavy chain variability field comprising sequence sequence identification number 12.

For the sake of clarity, Table 1 below summarizes the amino acid sequences of the present invention.

For those skilled in the art, any modification of the monoamino acid via an equivalent amino acid can be considered to be apparent and, therefore, part of the present invention.

Herein, the phrase "equal amino acid" is intended to indicate any amino acid which is likely to be substituted for one of the structural amino acids, however, it does not modify the biological activity of the corresponding antibody and is The biological activities of the particular instances are defined and, more particularly, the pI of the corresponding antibody is not modified. For example, an acidic amino acid can be substituted with another equivalent acidic amino acid.

In another complementary preferred embodiment of the invention, the variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, characterized in that the pI is comprised between 7 and 9, preferably Between 7 and 8.

As will be more apparent in the examples to those skilled in the art, the increase in pI of both the light and heavy chain variable regions has a significant impact on the homogeneity of Hz6F4-2.

This aspect of the invention is of particular interest, and thus a method is not merely unillustrated but also contrary to the teachings of the prior art.

In fact, prior art teaches that reducing pI would be an advantage in non-aggregation optimization (Arbabi-Ghahroudi & al., PEDS, Vol. 22, no. 2 pp59-66, 2009).

It is also known from prior art techniques to reduce pI, for example by designing variable regions, to reduce the elimination of IgG antibodies, in other words, to improve the half-life of antibodies (Igawa & al., PEDS, Vol. 23, no. 5 pp. 385-392, 2010).

The present invention clearly overcomes the prior art technical bias as it first suggests an increase in antibody pi.

According to yet another aspect, the present invention intends to variants of humanized antibodies, or the derived compounds or functional fragments, characterized in having a K _D JAM-A of between approximately 100 nM and about 100 pM in that. More preferably, the K _D JAM-A line of between about 50 nM and about 1 nM.

Wording "K _D" refer to a particular antibody-based - Solutions of antigen complex dissociation constant. K _D =K _off /K _on and K _off are composed of the "away rate" constant of the antibody dissociated from the antibody-antigen complex and the _Kon is composed of the level of the antibody-binding antigen (Chen Y. et al., 1999). J. Mol. Biol., 293: 865-881).

A novel aspect of the invention pertains to an isolated nucleic acid characterized in that it is selected among the following nucleic acids (including any degenerate genetic code):

a) a nucleic acid, DNA or RNA encoding an antibody, or a derivative or antigen-binding fragment thereof, according to the invention;

b) a nucleic acid comprising a heavy chain of the nucleic acid sequence sequence identification number 13 and the nucleic acid sequence sequence identification number 14, the sequence identification number 13 and the sequence identification number 14 each encoding the variant anthropomorphic having the sequence sequence identification number 11 The light chain of the antibody and the heavy chain of the variant anthropomorphic antibody having sequence identification number 12;

c) a nucleic acid complementary to a nucleic acid as defined in a) or b).

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleoside" are used interchangeably in this specification. "Acid sequence", meaning the precise sequence of a nucleotide, modified or not, defines a fragment or region of a nucleic acid, including non-natural nucleotides or not, and is a double strand of DNA, a single strand of DNA or Transcription products of such DNAs.

It should also be included herein that the present invention is not related to its natural chromosomal environment, that is, the nucleotide sequence in its natural state. The sequences of the invention have been isolated and/or purified, i.e., for example, by direct or indirect sampling by replication, the environment of which has been at least partially modified. By, for example, the recombinant nucleic acid obtained from the host cell, or the isolated nucleic acid obtained by chemical synthesis, should also be mentioned herein.

"A nuclear sequence that exhibits at least 80%, preferably 85%, 90%, 95%, and 98% percent identity with a preferred sequence after optimal alignment" means that the reference nuclear sequence is presented Certain modifications such as, in particular, censoring, truncation, extension, chimeric fusion and/or substitution, especially precise, nuclear sequences. Preferably, these are sequences encoding the same amino acid sequence as the reference sequence, which is a degeneracy of the genetic code, or a complementary sequence that is likely to specifically hybridize to the reference sequence, preferably Under highly stringent conditions, especially as defined below.

Hybridization under highly stringent conditions means that the conditions relating to temperature and ionic strength are selected such that hybridization between the two complementary DNA fragments is maintained in this manner. Based on merely exemplification, the highly stringent conditions of the hybridization step are advantageously as follows for the purpose of defining the polynucleotide fragments described above.

DNA-DNA or DNA-RNA hybridization was performed in two steps: (1) in a solution containing 5X SSC (1X SSC corresponding to 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% Pre-hybridization of sodium dodecyl sulfate (SDS), 10X Denhardt's, 5% dextran sulfate, and 1% salmon sperm DNA in phosphate buffer (20 mM, pH 7.5) at 42 °C (2) The main hybrid lasts for 20 hours at a temperature depending on the length of the probe (ie: 42 ° C for a probe >100 nucleotides in length) followed by 2 20-minute washes at 20 ° C in 2X SSC + 2% SDS, 1 time 20-minute wash at 20 ° C at 0.1X SSC + 0.1% SDS. The final wash was performed on 0.1X SSC + 0.1% SDS for 30 minutes at 60 ° C for a probe > 100 nucleotides in length. The highly stringent conditions described above for a defined size polynucleotide can be met by those skilled in the art, according to Sambrook, et al. (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd version, 2001). The procedures described therein were engineered for use with longer or shorter oligonucleotides.

The invention also relates to a vector comprising a nucleic acid as described in the present invention.

The invention is particularly directed to the selection and/or expression of vectors comprising such a nucleotide sequence.

The vector of the present invention preferably includes an element that allows the nucleotide sequence to be expressed and/or secreted in a putative host cell. The vector must therefore include a promoter, translation initiation and termination signals, as well as suitable transcriptional regulatory regions. It must be maintained in a host cell in a stable manner and can optionally have specific signals that specifically specify the secretion of the translated protein. These various elements are selected and optimized by the host cell skilled in the art. For this purpose, the nucleotide sequence can be inserted into a self-replicating vector in the host of choice or as an integrated vector for the host of choice.

Such vectors are prepared by methods well known to those skilled in the art and the pure strains produced can be introduced by standard methods, such as lipofection, electroporation, heat shock or chemical methods. Within a suitable host.

The vector is, for example, a plastid or viral derived vector. They are used to transform host cells to select or express the nucleotide sequences of the invention.

The invention also encompasses host cells which are transformed by a vector as described in the present invention or comprise a vector as described in the present invention.

The host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells, for example, but also yeast cells or animal cells, particularly mammalian cells. Insects or plant cells can also be used.

In addition to humans, the invention also relates to animals having cells transformed according to the invention.

Another aspect of the invention relates to a method of producing a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, of the invention, characterized in that the method comprises the steps of:

a) the culture in a medium of and the suitable culture conditions for a host cell according to the invention;

b) recovery of the anthropomorphic antibody, or derivative or antigen-binding fragment, of the variant produced from the culture medium or from the cultured cells.

The transfected cell line according to the present invention has a method for producing a recombinant polypeptide according to the present invention. Also included in the present invention are methods of preparing a polypeptide according to the recombinant form of the present invention, which are characterized by the use of a vector of the present invention and/or a cell transformed by a vector of the present invention. Preferably, a cell line transformed by a vector according to the invention is cultured under conditions which permit the performance of the aforementioned polypeptide and the recovery of the recombinant peptide.

As already mentioned, the host cell can be selected from prokaryotic or eukaryotic systems. In particular, it is possible to identify nucleotide sequences of the invention that would promote secretion in this prokaryotic or eukaryotic system. A vector carrying this sequence according to the invention can thus advantageously be used to produce a recombinant protein to be secreted. Rather, the fact that such recombinant proteins of interest are present within the supernatant of the cell culture rather than within the host cell promotes the purification of such recombinant proteins of interest.

The polypeptides of the invention may also be prepared by chemical synthesis. One such preparation method is also an object of the present invention. Those skilled in the art are aware of methods for chemical synthesis, such as solid phase techniques (see, in particular, Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed.) or particulate solid phase techniques. Condensation by fragmentation or by conventional synthesis in solution. Polypeptides obtained by chemical synthesis and polypeptides capable of containing corresponding unnatural amino acids are also included in the present invention.

Variant anthropomorphic antibodies, or derivatives or antigen-binding fragments thereof, which are likely to be obtained by the method of the present invention, are also included in the present invention.

In a preferred embodiment of the invention, the bispecific antibody antibody is a bivalent or tetravalent antibody.

Finally, the present invention relates to a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, as described above, for use as a medicament.

The present invention also relates to a composition comprising, as an active ingredient, a compound which is a humanized antibody of the variant of the present invention, or a derivative or antigen-binding fragment thereof. Preferably, the variant anthropomorphic antibody is supplemented with an excipient and/or a pharmaceutically acceptable carrier.

According to still another specific example, the present invention also relates to a pharmaceutical composition as described above, which comprises at least one second anticancer compound selected between the following compounds, which is capable of specifically inhibiting the receptor cheese Amino acid kinase activity, such as IGF-IR, EGFR, HER2/neu, cMET, VEGFR or VEGF, or any other anti-cancer compound known to those skilled in the art. In a second preferred aspect of the invention, the second compound can be selected between the following antibodies: anti-EGFR, anti-IGF-IR, anti-HER2/neu, anti-cMET, VEGFR, VEGF, etc. An isolated or functional fragment or derivative thereof that inhibits metastatic spread and/or anti-apoptosis and/or angiogenic and/or induced activity promoted by such receptors.

According to still another embodiment of the present invention, the composition further comprises, as a compound, at least one inhibitor of tyrosine kinase activity of a receptor such as IGF-IR, EGFR, HER2/neu, cMET and VEGFR. Products are available for simultaneous, separate or extended use.

In another preferred embodiment, the inhibitor of tyrosine kinase activity of the receptors is selected from the group consisting of a derivatized natural agent, diphenylamino quinone imine, pyrazole- or pyrrole-pyridinium or quinine. Group of oxazolines. Such inhibitors well known to those skilled in the art are described in the literature (Ciardiello F., Drugs 2000, Suppl. 1, 25-32).

Another embodiment complementary to the present invention consists of a composition as described above which additionally comprises a cytotoxic/cytostatic agent for use as a composite product for simultaneous, separate or extended means.

"Concurrent use" means the administration of two compounds comprising a composition in a single dosage form.

"Separate use" means the simultaneous administration of two compounds comprising the composition within the respective dosage form.

"Extended use" means the continuous administration of two compounds each comprising a composition within a respective dosage form.

In general, compositions in accordance with the present invention considerably increase the effectiveness of cancer treatment. In other words, the therapeutic effect of the antibodies of the invention is increased in an unexpected manner by administration of a cytotoxic agent. Another major subsequent advantage produced by the compositions of the present invention is that it is possible to use a lower effective amount of the active ingredient, thereby avoiding or reducing the risk of side effects, particularly for the action of cytotoxic agents, possibly of. Moreover, this composition makes it possible to achieve the desired therapeutic effect more quickly.

"Therapeutic anticancer agent" or "cytotoxic agent" means a substance that, when administered to a patient, treats or prevents the development of cancer in the patient. Non-limiting examples of such formulations include "alkylating" agents, antimetabolites, anticancer antibiotics, mitotic inhibitors, inhibitors of chromatin action, anti-angiogenic, anti-estrogen, antiandrogen, and immunomodulation Agent.

Such preparations, for example, are quoted in VIDAL as a dedicated page for compounds related to oncology and hematology according to the heading "Cytotoxicity"; the cytotoxic compounds cited in this document are cited in As a preferred cytotoxic agent herein.

In a particularly preferred embodiment, the composition of the invention as a composite product is characterized in that the cytotoxic agent is chemically bound to the variant anthropomorphic antibody for simultaneous use.

In a particularly preferred embodiment, the composition is characterized in that the cytotoxic/cytostatic agent is selected between a spindle inhibitor or a stabilizer, preferably vinorelbine and/or vinca fluoride. Vinflunine and / or vincristine.

In order to promote the binding between the cytotoxic agent and the anthropomorphic antibody according to the variant of the invention, a spacer molecule may be introduced between two compounds for binding, for example, a polyalkylene glycol polyethylene glycol or an amine group. The acid; or, in another embodiment, a reactive derivative of the cytotoxic agent can be used, and a function capable of reacting with the antibody has been introduced into the active derivative of the cytotoxic agent. Such combined techniques are well known to those skilled in the art and will not be discussed in greater detail in this specification.

Another aspect of the invention relates to a composition characterized in that at least one of the antibodies, or a derivative thereof or a functional fragment thereof, binds to a cell toxin and/or a radioisotope .

Preferably, the toxin or the radioisotope is capable of preventing the growth or proliferation of tumor cells, in particular not completely activating the tumor cells.

Moreover, preferably, the toxin is an enteric bacterial toxin, especially Pseudomonas exotoxin A.

The radioisotope which is preferably combined with the therapeutic antibody is a gamma ray-emitting radioisotope, preferably iodine ¹³¹ , 钇⁹⁰ , gold ¹⁹⁹ , palladium ¹⁰⁰ , copper ⁶⁷ , 铋²¹⁷ and 锑²¹¹ . Radioisotopes that emit alpha and beta rays can also be used in therapeutic methods.

"Toxin or radioisotope in combination with at least one antibody of the invention, or a functional fragment thereof," refers to any means by which binding of the toxin or the radioisotope to the at least one antibody is possible, especially by Covalent binding between two compounds, either introduction of the binding molecule or introduction of the binding molecule.

Examples of formulations which permit chemical (covalent), electrostatic, or non-covalent association of all or part of the binding element include, in particular, benzoquinone, carbodiimide and more particularly EDC (1-ethyl) -3-[3-dimethyl-aminopropyl]-carbodiimide-hydrochloride), dimethyleneimine, dithiobis-nitrobenzoic acid (DTNB) acid, N-amber N-succinimidyl S-acetyl thio-acetate (SATA), a bridging agent with one or more groups, with one or more stacks For the reaction of phenylaside groups with ultraviolet (UV) rays, the most preferred is N-[-4(azidoylylamino)butyl]-3'-(2'-pyridyldisulfide - Propylamine (APDP), N-succinimide-yl 3(2-pyridyldithio)propionate (SPDP) and 6-mercapto-nicotinamide (HYNIC).

Another form of combination, especially with respect to radioisotopes, can be made up of the use of bifunctional ion chelating agents.

Examples of such chelating agents include chelating agents derived from EDTA (ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaacetic acid), which are developed to bind metals, particularly radioactive metals, with immunoglobulins. Protein together. Thus, DTPA and its derivatives can be substituted on the carbon chain by a variety of groups, thereby increasing the stability and rigidity of the ligand-metal complex (Krejcarek et al., 1977; Brechbiel et al., 1991; Gansow, 1991; U.S. Patent 4,831,175).

For example, DTPA (diethylenetriamine pentaacetic acid) and its derivatives, etc., have been widely used in medicines and biology for a long time, either in their free form or with a metal ion. a form of a complex that exhibits a distinct characteristic of a stable chelate with a metal ion, which can be coupled to a protein having therapeutic or diagnostic benefits, such as an antibody, for use in the development of a radioimmunoconjugate for cancer therapy. (Meases et al., 1984; Gansow et al., 1990).

Moreover, preferably, at least one of the anti-systems of the invention which form the conjugate is selected between its functional fragments, particularly fragments which have lost their Fc components, such as scFv fragments.

The invention also encompasses the use of the composition for the preparation of a medicament, preferably intended for the prevention or treatment of cancer.

The invention also relates to the use of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, and/or the use of a composition according to the invention for the preparation of a medicament for inhibiting the growth of tumor cells. In general, the invention relates to the use of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, and/or a composition for the manufacture of a medicament for the prophylaxis or treatment of cancer.

The invention also relates to the use of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, and/or the use of a composition according to the invention for the preparation of a medicament for inhibiting the growth of tumor cells. In general, the present invention relates to the use of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, and/or a composition for the preparation of a medicament for the prevention or treatment of a disease associated with tumor cell proliferation. The use of the object.

Preferred cancers that can be prevented and/or treated include prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, colon cancer, multiple myeloma, ovarian cancer, pancreatic cancer, or any other cancer.

In a preferred form, the cancer is a cancer selected between estrogen-related breast cancer, non-small cell lung cancer, colon cancer, and/or pancreatic cancer.

Another aspect of the invention pertains to a method of diagnosing, preferably in vitro, one of the diseases associated with the variant anthropomorphic antibody as described in the JAM-A performance level. Preferably, the JAM-A protein related disease in the diagnostic method will be cancer.

Thus, the variant anthropomorphic antibody of the present invention, or any derivative or antigen-binding fragment, can be used in a method for detecting and/or quantifying JAM-A protein in a biological sample in vitro, especially For the diagnosis of a disease associated with the abnormal performance of this protein, such as cancer, wherein the method comprises the following steps:

a) placing the biological sample in contact with a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, of the invention;

b) exhibit antigen-antibody complexes that may form.

Thus, the present invention also encompasses a kit or accessory for implementing a method as illustrated, comprising the steps of:

a) a variant anthropomorphic antibody of the invention;

b) optionally, an agent for constituting a medium that is advantageous for the immune response;

c) Optionally, an agent that exhibits an antigen-antibody complex produced by an immune response.

Advantageously, the variant anthropomorphic antibody, or antigen-binding fragment thereof, can be immobilized on a support, especially a protein wafer. One such protein wafer is an object of the present invention.

Advantageously, the protein wafer can be used in kits or accessories required for detecting and/or quantifying the JAM-A protein in a biological sample.

It must be stated that the term "biological sample" is used herein to refer to a sample taken from a living organism (especially blood, tissue, organs or other samples taken from a mammal, especially a human) or is likely to contain Any sample of the JAM-A protein (eg, a sample of cells, if desired, is a sample of transformed cells).

The variant anthropomorphic antibody can be in the form of an immunoconjugate or a labeled antibody to obtain a detectable signal and/or a measurable signal.

The labeled antibody of the present invention, or a function or fragment thereof, includes, for example, an antibody conjugate (immunoconjugate), which may be combined, for example, with an enzyme such as a peroxidase or a base. Phosphatase, α-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenation The enzyme is either by a molecule such as biotin, longleaf foxglove or 5-bromo-deoxyuridine. Fluorescent labels can also be combined with functional fragments of the antibodies of the invention or the like, including, in particular, fluorescent yellow and its derivatives, fluorescent dyes, rose bengal and its derivatives, green fluorescent protein (GFP), Dansyl, umbelliferone, and the like. In such combinations, the antibody of the invention or a functional fragment thereof, etc., can be prepared by methods known to those skilled in the art. These may be directly bound by enzyme or fluorescent label; via a spacer group or a linking group, such as polyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) or diethylenetriamine Acetic acid (DPTA); or in the presence of a binder, such as those mentioned above for therapeutic conjugates. The conjugate carrying the fluorescent yellow label can be prepared by reaction with isothiocyanate.

Other conjugates may also include chemiluminescent labels such as luminal and dioxetane, bioluminescent labels such as luciferase and luciferin, or radioactive labels. For example, iodine ¹²³ , iodine ¹²⁵ , iodine ¹²⁶ , iodine ¹³³ , bromine ⁷⁷ , 鎝^99m , indium ¹¹¹ , indium ^113m , gallium ⁶⁷ , gallium ⁶⁸ , 钌⁹⁵ , 钌⁹⁷ , 钌¹⁰³ , 钌¹⁰⁵ , mercury ¹⁰⁷ , mercury ²⁰³ ,铼^99m , 铼¹⁰¹ , 铼¹⁰⁵ , 钪⁴⁷ , 碲^121m , 碲^122m , 碲^125m , 銩¹⁶⁵ , 銩¹⁶⁷ , 銩¹⁶⁸ , fluorine ¹⁸ , 钇¹⁹⁹ and iodine ¹³¹ . Existing methods known to those skilled in the art for combining radioisotopes with antibodies, whether directly or via a chelating agent, such as the EDTA or DTPA mentioned above, can be used as diagnostic radioisotopes. Thus, reference should be made to the labeling of [I ¹²⁵ ]Na [Hunter WM and Greenwood FC (1962) Nature 194:495] by the technique of chloramine-T; as indicated by Crockford et al., labeled with 鎝^99m (US patent) 4,424,200) or associated via DTPA as illustrated by Hnatowich (U.S. Patent 4,479,930).

The invention also relates to the use of a variant anthropomorphic antibody according to the invention for the preparation of a medicament for the specific alignment of a compound which is biologically active for cellular expression or overexpression of the JAM-A protein.

As used herein, a "biologically active compound" is any compound that is capable of modulating, particularly inhibiting, cell activity, particularly growth, proliferation, transcription, and gene translation.

The invention also relates to an in vivo diagnostic reagent consisting of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, of the invention, preferably labeled, in particular radiolabeled And its use in medical imaging, especially for detecting cancer associated with cellular performance or overexpression of JAM-A protein.

The invention also relates to a composition as a composite product or to an anti-JAM-A/toxin conjugate or radioisotope, for use as a medicament in accordance with the invention.

Preferably, the composition or the combination as a composite product will be supplemented with an excipient and/or a pharmaceutically acceptable vehicle.

In the present specification, "pharmaceutical vehicle" means a compound added as a pharmaceutical composition, or a combination of compounds, which does not cause secondary reactions and, for example, facilitates administration of the active compound, and increases in the organism. The full life and/or effectiveness of the life increases its solubility in the solution or improves its storage. Such pharmaceutically acceptable carriers are well known and will be modified by a person skilled in the art depending upon the nature of the active compound selected and the route of administration.

Preferably, such compounds are administered by a systemic route, particularly by intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or oral routes. More preferably, the composition consisting of the variant anthropomorphic antibodies according to the invention will be administered in a number of doses that are evenly spaced over time.

When a treatment suitable for a patient is established, its route of administration, schedule of administration, and optimal form of formulation can be determined according to generally considered criteria, such as, for example, the age or weight of the patient, his general State severity, his treatment tolerance, and the side effects experienced.

Thus, the present invention relates to the use of a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, for the preparation of a medicament for the specific alignment of a compound which is biologically active for cellular expression or overexpression of JAM-A of.

Other features and advantages of the present invention are further disclosed in the description and the examples and illustrations, which are set forth below.

Graphical description

Figure 1 shows the alignment between mouse 6F4 VH and selected human V-genes and J-genes; Figure 2 shows mouse 6F4 VH and selected human V-genes Arrangement with each of the J-genes; Figure 3 illustrates the binding of JAM-A ECD by ELISA; chimeric and anthropomorphic antibodies (A: heavy chain; B: light chain) to JAM-A cells The binding activity of the outer domain (ECD) is measured by ELISA, where an anti-human Cκ conjugate is used to detect purified antibodies, dose-dependent binding activity to the plastic coated JAM-A ECD Measured at 450 nm; Figure 4 illustrates the competitive binding of biotinylated murine 6F4 to JAM-A ECD by ELISA; via chimeric and anthropomorphic (A: heavy chain; B: light chain) antibodies Competitive binding to the JAM-A extracellular domain (ECD) of biotinylated murine 6F4 Mab was measured by ELISA; dose-dependent replacement of biotinylated 6F4 to plastic coated JAM-A ECD Monitored above; Figure 5 illustrates competitive binding of antibody binding by JAM-A ECD by ELISA: anthropomorphic heavy chain and anthropomorphic light chain; chimeric and anthropomorphic M The binding activity of ab to the JAM-A extracellular domain (ECD) (A) was measured by ELISA using an anti-human CK conjugate; the biotinylated murine 6F4 Mab was via a chimeric and anthropomorphic antibody ( B) Competitive binding to the JAM-A extracellular domain (ECD) was measured by ELISA; Figure 6 shows the IEF gel for Hz6F4-1 vs. Hz6F4-2 (2 batches); 7A and 7B The figure shows capillary isoelectric focusing (cIEF) for Hz6F4-1(A) and Hz6F4-2(B); Figures 8A and 8B show cation exchange for Hz6F4-1(A) and Hz6F4-2(B) Chromatography (CEX); Figures 9A and 9B show the results of mass spectrometry for the entire antibody (IgG) of Hz6F4-1 (A) and Hz6F4-2 (B); Figures 10A and 10B show Hz6F4- 1 (A) and Hz6F4-2 (B) results of mass spectrometry of whole antibodies subjected to PNGase F digestion (IgG); Figures 11A and 11B show Hz6F4-1 (A) and Hz6F4-2 (B) Results of mass spectrometry of heavy chain (HC); Figures 12A and 12B show the results of mass spectrometry of heavy chain (HC) digested with PNGase F for Hz6F4-1 (A) and Hz6F4-2 (B); Figure 13 corresponds to the A-431 xenograft tumor model; Figure 14 corresponds to the DU-145 xenograft tumor model; Figure 15 illustrates Inhibition of proliferation of A431 cells by Mab Hz6F4-2; Figures 16A, B and C illustrate downward regulation of JAM-A by Mab Hz6F4-2 using A: Western blot, B: densitometry And C: immunofluorescence staining; Figure 17 illustrates the effect of Mab Hz6F4-2 on the expression of adhesive molecules by western blots; Figure 18 illustrates the in vivo activity of hz6F4-2 Mab on the A431 xenograft model.

Example Example 1: Anthropomorphic strategy for backup candidates for 6F4 antibodies

In the first step of the anthropomorphization of mouse 6F4 monoclonal antibody (Mab), a chimeric form is fused to human CK and human IGHG4 by the mouse variability domain, m6F4-VH and m6F4-VL, respectively. The conservation field is to be constructed. The chimeric antibodies produced belong to the human IgG4/κ subtype.

In the next step, the first anthropomorphic form of 6F4 Mab was designed based on the amino acid sequence homology to select the human reproductive V-gene as close as possible to the mouse V-gene. By applying this criterion, IGHV1-f*01 and IGKV1-33*01 were each selected for acceptor sequences of heavy and light chains. However, the anthropomorphic Mab produced by hz6F4-V1 produced a calculated isoelectric point (pI) of 5.96 (calculated using the 'iep' application from the wEMBOSS v5.0.0 software), which makes this Mab not It is easy to process by a conventional upstream method purification platform, in particular with respect to the viral inactivation step performed at acidic pH.

Thus, a novel anthropomorphic strategy is designed in which the first selection criterion for human V-gene acceptor sequences is not exclusively based on amino acid sequence homogeneity, but on the most basic pI, and preferentially The pI values above 7.0 of the V-gene were received for each VH and VL. The definition of the framework and CDR domain and the selection of the human recipient reproductive V-gene are performed using the tools and libraries of the IMGT website (www.imgt.org). Similarly, the amino acid residues containing a basic side chain (ie, arginine, lysine, histidine) will be more conserved during the evaluation of the putative backup mutations in the anthropomorphic design. And do not think it will be back-mutations.

Estimates of the calculated pI values for each anthropomorphic, CDR-transplanted Mab are performed using the 'iep' application from the wEMBOSS v5.0.0 software.

The anthropomorphic variant can be cloned into a pConPlus vector that does not carry a Cκ conservation domain (pConPlusKappa2, Lonza Biologics, Slough, UK) or an IGHG4 conservation domain (pConPlusGamma4PRODeltaK, Lonza Biologics, Slough, UK) And temporarily expressed in CHO-K1SV cells. Their ability to bind JAM-A target proteins will be assessed by ELISA and compared to a chimeric format of 6F4 Mab as detailed in the examples below.

a) Design of anthropomorphic 6F4 backup candidates for heavy chain variable field VH

- Selection of human acceptor V-gene and J-gene

An IGHV1-f*01V-gene was searched for the best human hit using the IMGT V-quest tool and a traditional database search based on the nucleotide sequence of the mouse 6F4 VH domain. This human reproduction series V-gene produces a low pI value of 6.25 and is not selected. In the next step, we applied a database search based on the IMGT DomainGapAlign tool and the amino acid sequence in the mouse 6F4 VH domain.

- theoretical, CDR-transplanted anthropomorphic form of pI calculation

This calculation was performed by reconstituting the full-length heavy chain corresponding to the theory of the fully anthropomorphic form (each human V-gene carrying three CDRs of mouse 6F4 and one human IGHJ4*01J-gene, and one IgG4) Conservation field)

Based on the pI calculation and sequence homogeneity, the selected acceptor human V-gene and J-gene are IGHV1-3*01 and IGHJ4*01.

Figure 1 shows the alignment between mouse 6F4 VH and each of the selected human V-genes and J-genes.

The human IGHV1-3*01 gene has 28 amino acid differences from mouse 6F4 VH, and 2 additional J-regions.

Each of these locations will be analyzed separately taking into account several physical-chemical and structural parameters:

● The isoelectric point of the amino acid. Thirteen of the 30 residues are involved in charged amino acids (whether alkaline or acidic). Basic residues, such as lysine, histidine or arginine, are kept as conserved as possible to maintain an overall pI above 7.0;

● The position of the fixture as a CDRs will be conserved as a mouse in the first instance;

● The cursor zero residue is considered to be high priority.

According to the above mentioned criteria, an anthropomorphic form of Hz6F4-2 VH is designed:

Of the 30 different residues between the mouse donor and human acceptor sequences, 21 were directly introduced as human residues and were not evaluated as back mutations, due to their non-criticality in terms of VH structure. s position. These also facilitate the introduction of basic residues such as Lys12, His35 and Arg87.

b) Design of anthropomorphic 6F4 backup candidates for light chain variability field VL

- Selection of human acceptor V-gene and J-gene

A novel library search based on the IMGT V-quest tool and a nucleotide sequence based on the nucleotide sequence of the mouse 6F4 VH. The novel putative human reproductive series V-gene (best hit) listed below and its Equal to the percent identity of both the nucleotide and amino acid levels.

Note: The IGKV1-33V-gene was selected as the human acceptor sequence of the first anthropomorphic strategy. It is not selected for the design described below. A list of the closest human IGKV genes that are identical based on the V-gene rating of the DomainGapAlign tool.

- theoretical, CDR-transplanted anthropomorphic form of pI calculation

This calculation is performed by reconstituting the full-length light chain corresponding to the theory of the fully anthropomorphic form (each human V-gene carrying three CDRs of mouse 6F4 and one human IGKJ1*01J-gene, and one human) Cκ conservation field)

Based on the pI calculation and sequence homogeneity, the selected acceptor human V-gene and J-gene are IGKV1D-43*01 and IGKJ1*01.

Figure 2 shows the alignment between mouse 6F4 VH and each of the selected human V-genes and J-genes.

The human IGKV1D-43*01 gene has 25 amino acid differences from mouse 6F4 VL, and 2 additional J-regions.

Each of these locations will be analyzed separately taking into account several physical-chemical and structural parameters:

● The isoelectric point of the amino acid. The 8 residues relate to charged amino acids (whether alkaline or acidic). Basic residues, such as lysine, histidine or arginine, are kept as conserved as possible to maintain an overall pI above 7.0;

● The position of the fixture as a CDRs will be conserved as a mouse in the first instance;

● The cursor zero residue is considered to be high priority.

According to the above mentioned criteria, an anthropomorphic form of Hz6F4-2 VL was designed.

Of the 27 different residues between the mouse donor and human acceptor sequences, 21 were directly introduced as human residues and were not evaluated as back mutations, due to their non-criticality in terms of VL structure. s position. The CDR2 retainers His49 and Thr53 are conserved counterparts of their counterparts, since they are equal to the strong involvement of the CDR structure and the basic side chain (His).

real Example 2: Production of chimeric and anthropomorphic 6F4 Mabs

All Mab lines were produced after transient transfection and by using the CHO-K1 SV system with expression vectors (Lonza Biologics, Slough, UK) with pConPlusKappa2 (light chain) and pConPlusGamma4PRODeltaK (heavy chain).

The entire nucleotide sequence corresponding to the anthropomorphic form of the variable domain of the 6F4 Mab light and heavy chains was synthesized by whole gene synthesis (Genecust, Luxembourg). They are sub-selected into the expression vector (Lonza Biologics, Slough, UK) carrying the entire coding sequence of the corresponding conservation domain, pConPlusKappa2 (light chain) or pConPlusGamma4PRODeltaK (heavy chain). All selection steps were performed according to conventional molecular biology techniques as described in the Laboratory manual (Sambrook and Russel, 2001) or according to the supplier's guidelines. Each gene construct was completely confirmed by nucleotide sequencing using a Big Dye terminator cycle sequencing kit (Applied Biosystems, US) and analyzed using a 3100 Genetic Analyzer (Applied Biosystems, US).

Suspension-modified CHO-K1SV cells (Lonza Biologics, Slough) were routinely grown on an orbital shaker in 50 ml of chemically defined serum-free CD CHO medium (InVitrogen, US) at 250 ml Among the flasks. Transient transfections were performed by electroporation using a GenePulser instrument (BioRad Laboratories, US) in a volume of 600 μl and an 80 μg expression vector of 10 ⁷ cells and a 2:1 ratio H/L chain vector. The transfection mixture was used to inoculate 30-50 ml of culture in CD-CHO medium. The culture method is monitored based on cell viability and Mab production. Typically, the culture is maintained for 5 to 7 days. Mabs were purified on Protein A resin (GE Healthcare, US) using conventional chromatographic methods.

Antibody formats are produced in a level suitable for ELISA evaluation. Productivity levels typically range between 1 and 5 mg/l of purified Mabs.

Example 3: Evaluation of the binding ability of JAM-A by chimeric and anthropomorphic Mabs by ELISA

The first screening assay corresponds to the ability of chimeric and anthropomorphic Mabs to bind to the extracellular domain of JAM-A.

Briefly, ELISA plates (Immulon II HB, VWR) were coated with recombinant protein [4 μg/ml] corresponding to the JAM-A extracellular domain overnight at 4 ° C and produced in E. coli and in PBS. Saturated with 1% bovine serum albumin (BSA). After 3 washes with PBS containing 0.05% Tween 20, the samples to be analyzed (200 μl) were serially diluted, added to a microtiter plate and incubated at 37 °C for 1H. After 3 washes, the anti-human C-kappa antibody (Ref. A7164, Sigma Aldrich) coupled to peroxidase was added at 1/5000 dilution and incubation at 37 °C for 1 H. . After 3 additional washes, a peroxidase colorimetric substrate (ELISA peroxidase substrate, Interchim) was added and incubation at room temperature for 5 min.

Figure 3A shows that the binding ability of the anthropomorphic heavy chain Hz6F4-2 VH combined with a chimeric 6F4 Mab light chain is almost identical to that of a chimeric 6F4 Mab. Similar results were obtained with the anthropomorphic light chain Hz6F4-2 VL combined with a chimeric heavy chain (Fig. 3B).

Example 4: Evaluation of the ability of anthropomorphic Mabs to compete with biotinylated murine 6F4 Mab for binding to JAM-A

The second screening assay corresponds to the ability of chimeric and anthropomorphic Mabs to compete with biotinylated mouse 6F4 Mab for binding to the JAM-A extracellular domain.

* 6F4 Mab biotinylation procedure : 6F4 Mab is labeled with biotin (sulfonate-NHS-LC-biotin, Perbio Science-1 mg/ml at 20/l, mol: mol of 0.1 M sodium bicarbonate The buffer-dye-to-antibody ratio) and the mixture were incubated in the dark for 30 min at room temperature. The reaction was immediately dialyzed against PBS overnight at 4 ° C in the dark.

The concentration of the labeled Mab protein is determined by BCA analysis. It is also characterized by SDS-PAGE electrophoresis under reducing and non-reducing conditions. No difference was observed with the unlabeled m6F4 Mab. The labeling of biotin was further confirmed by the western blot of the evaluated: the integrity of the labeled Mab solution and showed that biotin was equally coupled to both the heavy and light chains.

* ELISA procedure : ELISA plate (Immulon II HB, VWR) was coated with recombinant protein [4 μg/ml] corresponding to the JAM-A extracellular domain overnight at 4 ° C and produced in E. coli and in PBS. Saturated with 1% bovine serum albumin (BSA). After 3 washes with PBS containing 0.05% Tween 20, the samples to be analyzed (200 μl) were serially diluted and together with a fixed concentration of biotinylated mouse 6F4 Mab [biot-m6F4: 2 μg/ml] Add to the microtiter plate. Incubation was performed at 37 ° C for 1 H. After 3 washes, the detection system corresponding to avidin-coupled peroxidase (Ref. A3151-1MG, Sigma Aldrich) was added at a dilution of 1/2000 and incubated at 37 °C for 1 H. . After 3 additional washes, a peroxidase colorimetric substrate (ELISA peroxidase substrate, Interchim) was added and incubation at room temperature for 5 min.

Figure 2 shows the competitive binding ability of the anthropomorphic heavy chain to the light chain in combination with a chimeric 6F4 Mab counterpart. Hz6F4-2 VH (Fig. 4A) and Hz6F4-2 VL (Fig. 4B) produce potent and efficient ability to inhibit the binding of biot-m6F4 to JAM-A ECD.

Example 5: Evaluation of the ability of anthropomorphic Mabs to compete with biotinylated murine 6F4 Mab for binding to JAM-A

The combination of the Hz6F4-2 VH line and the Hz6F4-2 VL chain and the resulting resistance system evaluates its ability to bind JAM-A ECD and its ability to replace bio-m6F4 with its target antigen, as illustrated in the above examples. of.

Figure 5A shows the ability of a fully anthropomorphic Mab to recognize JAM-A ECD. Hz6F4-2 produced a binding capacity of the extracellular domain of JAM-A which is almost identical to a chimeric 6F4 Mab.

Figure 5B shows the fully anthropomorphic Mab to inhibit the competitive binding ability of biot-m6F4 binding to JAM-A ECD. Hz6F4-2 is a powerful and effective competitor for biot-m6F4 binding to JAM-A ECD.

Embodiment 6: Isoelectric focusing (IEF): Hz6F4-1 and Hz6F4-2

Samples were analyzed by IEF (3-10) on an AMERSHAM Multiphor II electrophoresis unit.

The dried gel was rehydrated with 16.5 ml of buffer prepared from 1.65 g of sorbitol, 1230 μl of pharmalytes pH 3-10 and water. After sonication, the reconstituted aqueous solution is placed within the GelPool and the gel is placed down on the surface. It can be removed and used immediately after 2 hours.

The Multiphor II electrophoresis unit was connected to a cryostat and the temperature was set to 10 °C for 15 minutes before starting the analysis. Place 5 ml of glycerol anhydre onto the cooling plate of the Multiphor II and place the gel in place, taking care not to catch air bubbles underneath.

Place the sample application slide in place and place a 15 μl (5 μg) sample concentration of 0.33 mg/ml and a pI 5-10.5 15 μl standard.

The migration was performed for 3 hours, 0V to 2000V, and paused during the break to remove the sample application sheet. The gel was colored with Coomassie Blue and dried with cellophane. Scan the gel and use the chip (logicity "Quantity One" to determine the pI.

As illustrated by Figure 6, the obtained Hz6F4-1 vs. Hz6F4-2 IEF gel (2 batches) both showed a pI increase of approximately 2 units (5.8 vs. 7.7) and a significantly reduced number of Hz6F4-2 band.

Example 7: Capillary isoelectric focusing (cIEF): Hz6F4-1 and Hz6F4-2

A feasibility study was carried out by BECKMAN using Hz6F4-1 and Hz6F4-2. The isoelectric point (pI) assay was performed on a ProteomLab PA800 from BECKMAN instrument. A 30 cm long and 50 μm I.D. diameter eCAP neutral capillary was used at a temperature of 20 ° C and a pore size of 100 x 200 μm.

Preparation of analyte (200 mM phosphoric acid), catholyte (sodium hydroxide 300 mM), chemical activator (350 mM acetic acid), catal stabilizer (L-arginine, 500 mM), anode stabilizer (imine diacetic acid, 200 mM), urea solution (urea, 4, 3M) and 3M urea-cIEF gel solution.

The analyte solution consisted of 200 mM H3PO4 in water. The catholyte solution consisted of 300 mM NaOH in water. The chemical activator consisted of 350 mM CH ₃ COOH in water. The catal stabilizer consisted of 500 mM L-arginine in water. The anode stabilizer consisted of 200 mM imine diacetic acid in water. The 3M urea-cIEF gel solution consisted of urea 3M in the cIEF gel kit.

The urea solution was used at 50 psi for a period of 5 minutes to flush the capillary between analyses.

One main mixed solution is 200 μL of 3M urea-cIEF gel, 12 μL of ampholyte pH 3-10 (GE Healthcare, ref 17-0456), 20 μL of catal stabilizer, 2 μL of anode stabilizer, and pI The label (2 μl of peptide pI 10; 2 μl of peptide pI 9, 5 and 2 μL of peptide pI 4,1) was prepared.

The protein sample was buffer exchanged with Tris 20 mM solution on microcon YM-30 because the protein sample must have a salt concentration below 50 mM.

After rinsing the capillary with a 4,3 mM urea solution and H2O solution for 2 min, the sample was injected to the anode side. A focusing step is performed at 25 kV during 15 min and then at 30 kV during 30 min. Detection was performed at 280 nm and data was analyzed using Beckman 32 Karat 5.0 software.

Figures 7A and 7B show data from the cIEF experiment. Again Hz6F4-2 is clearly more homogenous than Hz6F4-1.

Example 8: Cation exchange chromatography: Hz6F4-1 and Hz6F4-2

a) Cation exchange chromatography: Hz6F4-1 (Fig. 8A)

Hz6F4-1 can be formulated in different buffers, for example, 20 mM His; 150 mM NaCl pH 6.0; 0.5% (w/w) Tween 80.

The different homogeneous systems of Hz6F4-1 were separated on a Dionex WCX-10 cation exchange column on a Waters HPLC system, 4 x 250 mm and a WCX-10 protection column 4 x 50 mm. It consists of a 515-barrel, a 717 auto-injector and a 2487 UV detector. Buffer A was 20 mM sodium acetate, pH 5.0, and buffer B was 20 mM sodium acetate with 1 M NaCl, pH 5.0. The flow rate was 1.0 ml/min. The amount injected is approximately 20 μg. The gradient used for elution was 0 min with 100% solvent A followed by a linear gradient of 25% solvent B during 77.5 min. After elution, the column was washed with 75% solvent B for 9 min and re-equilibrated with 100% solvent A for 25 min. The elution was monitored by UV spectroscopy at 280 nm.

b) cation exchange chromatography: Hz6F4-2 (Fig. 8B )

Hz6F4-2 was formulated in a different buffer, for example, 35 mM phosphate 75 mM NaCl at pH 6.2.

The different homogeneous systems of Hz6F4-2 were separated on a Dionex WCX-10 cation exchange column on a Waters HPLC system, 4 x 250 mm and a WCX-10 protection column 4 x 50 mm. It consists of a 515-barrel, a 717 auto-injector and a 2487 UV detector. Buffer A was 20 mM sodium acetate, pH 6.0, and buffer B was 20 mM disodium hydrogen phosphate with 1 M NaCl, pH 6.0. The flow rate was 1.0 ml/min. The amount injected is approximately 20 μg. The gradient used for elution was 0 min with 4% solvent B followed by a linear gradient of 4-15% solvent B during 73 min. After elution, the column was washed with 75% solvent B for 9 min and re-equilibrated with 96% solvent A for 25 min. The elution was monitored by UV spectroscopy at 280 nm.

Figures 8A and 8B show data from CEX analysis. Hz6F4-2 is obviously more homogenous than Hz6F4-1.

Example 9: Mass Spectrometry: No PNGase F Treatment and Whole Antibody with PNGase F Treatment

- Equipment :

● LCT Premier Waters (SM04) mass spectrometer equipped with Masslynx ^TM 4.1, Transform ^TM 4.1, MaxEnt ^TM 4.1, Biolynx ^TM 4.1

● Alliance HPLC (CH07)

● UV detector (PDA) DT 0003

● Desalting column: Mass Prep Online Desalting Cartridge (2.1x10 mm, Waters)

● 2 mL vial (Waters, WAT270946C) and 150 μL insert (Waters, ref: WAT094171DV)

● 250 μL syringe (Hamilton, GasTight 1725RNR, ref: 81165)

● 0.5 mL centrifuge tube

● Straw and matching pipette head

● Thermostatic mixer + plug tube 0.5 mL

-Chemicals/Reagents :

● Preparation of HPLC elution solution:

o Acetonitrile (Carlo Erba, ref P00637S21 or similar)

o Formic acid (FA, Sigma/Fluka, ref: 06440 or similar)

o MilliQ water

● Isopropanol (Isopropylic Alcohol, Carlo Erba, ref: 412712 or similar)

● Peptide N-glycosidase (PNGase F, BioLabs New Engl and ref: P0704L/75 000 units or similar)

● cesium iodide (CsI, Merck, ref: 102861 or similar)

● Reference mAb: B6AHM or similar

- Preparation of samples :

Complete non-deglysylated mAbs were injected without any prior treatment. In terms of analysis, one sample volume was taken to have 15 μg and 10 μg was injected. For LCMS analysis, a desalting column Mass Prep Online Desalting Cartridge (2.1 x 10 mm, Waters) and a flow rate of 0.4 mL/min were used. The HPLC elution solution was acetonitrile 0.1% formic acid for solvent B and water 0.1% formic acid for solvent A. The flow rate was 0.4 mL/min and the oven temperature was 60 °C. The gradient used for elution was 1 min in 95% solvent A, 3 min 30 in 80% solvent B and 2 min 30 in 95% solvent A. Mass spectrometry was performed on top of the LCT Premier with a 3400V capillary, desolvation temperature of 350 ° C, source temperature of 150 V and a sample cone of 120 V.

For the deglycosylated sample, 100 μg of Mab was obtained in a 0.5 mL centrifuge tube. 0.5 μL of PNGase F was added and incubated overnight at 37 ° C in a thermomixer and mixed (800 t/min). In terms of analysis, one sample volume was taken to have 15 μg and 10 μg was injected. For LCMS analysis, a desalting column Mass Prep Online Desalting Cartridge (2.1 x 10 mm, Waters) and a flow rate of 0.4 mL/min were used. The HPLC elution solution was acetonitrile 0.1% formic acid for solvent B and water 0.1% formic acid for solvent A. The flow rate was 0.4 mL/min and the oven temperature was 60 °C. The gradient used for elution was 1 min at 95% solvent A, 3 min 30 at 80% solvent B and 2 min 30 at 95% solvent A. Mass spectrometry was performed on top of LCT Premier (Micromass Waters) with a 3,400 V capillary, desolvation temperature of 350 ° C, source temperature of 150 V and a sample cone of 120 V.

- Results:

For the entire antibody (IgG), Figures 9A and 9B show data for LC-ESI-TOF experiments. Hz6F4-2 (Fig. 9B) is clearly more homogenous than Hz6F4-1 (Fig. 9A). Hz6F4-2 shows four major peaks corresponding to the expected IgG+ major glycoforms (eg, G0/G0F, G0F/G1F, G1F/G1F, G1F/G2F). Hz6F4-1 also showed four major peaks corresponding to the expected IgG+ glycoforms (eg, G0/G0F, G0F/G1F, G1F/G1F, G1F/G2F), but with high amounts of additional impurities.

Regarding the entire antibody (IgG) subjected to PNGase F digestion, FIGS. 10A and 10B show data of the LC-ESI-TOF experiment. Hz6F4-2 (Fig. 10B) is clearly more homogenous than Hz6F4-1 (Fig. 10A). After PNGase F digestion, Hz6F4-2 showed a major peak corresponding to the expected non-deglysylated IgG, and a minor peak was also seen, which may correspond to two glycation sites (+340 Da). Conversely, Hz6F4-1 also shows a major peak corresponding to the expected deglycosylated IgG, but with a very high amount of at least 5 additional impurities of higher quality.

Example 10: Mass Spectrometry: Antibodies without PNGase F treatment and reduced with PNGase F treatment (light and heavy chains)

- Equipment:

● LCT Premier Waters (SM04) mass spectrometer equipped with Masslynx ^TM 4.1, Transform ^TM 4.1, MaxEnt ^TM 4.1, Biolynx ^TM 4.1

● Alliance HPLC (CH07)

● UV detector (PDA) (DT 0003)

● Column X BridgeBEH 300 C4 2,1 x 150 mm,3,5 μm (Waters PN 186004500 LN 0104191601)

• 2 mL vial (Waters, WAT270946C) and 150 μL insert (Waters, ref WAT094171DV)

● 250 μL syringe (Hamilton, GasTight 1725RNR, ref 81165)

● 0.5 mL centrifuge tube

● Straw and matching pipette head

● Thermostatic mixer + plug tube 0,5 mL

● Freeze dryer (LY01).

-Chemicals/Reagents:

● Preparation of HPLC elution solution:

o n-propanol (n-propyl alcohol, Carlo-Erba, ref: 412542 or similar)

o Formic acid (FA, Sigma/Fluka, ref: 06440 or similar)

o Trifluoroacetic acid (TFA, Fluka, ref: 91699 or similar)

o MilliQ water

● Preparation of reduction buffer:

o Tris HCl (Trizma Base, Sigma, ref: T6066 or similar)

o Ethylenediaminetetraacetic acid (EDTA, ref: ED2SS or similar)

o Guanidine chlorhydrate (Guanidine hydrochlorid 99%, Aldrich, ref: 177253 or similar)

o MilliQ water

o Hydrochloric acid 6N (HCl, Merck, ref: 100314 or similar)

o Dithiothreitol (DTT, Aldrich, ref: 150460 or similar)

● Isopropanol (Isopropylic Alcohol, Carlo Erba, ref: 412712 or similar)

- Preparation of samples:

For the non-deglycosylated reducing mAb, a sample volume corresponding to 100 μg of mAb was freeze-dried in a 0.5 mL centrifuge tube. Dissolved in 20 μl of reducing buffer (0.121 g Tris HCl, 7.4 mg EDTA, 5.73 g guanidine HCl, dissolved in 9 ml with MilliQ water, adjusted to pH 8.0 with HCL 6N and complete to 10 mL with MilliQ water). Under a nitrogen flow of 30 s, 0.8 μL of a 80 mg/mL DTT solution was added and, under mixing (800 t/min), 2H was incubated at 37 ° C in a thermostat mixer. The reaction was quenched by the addition of 2 μL of acetic acid and 4 μL of n-propanol. Add 73.2 μL of MilliQ water just prior to injection.

For the deglycosylated mAb, one sample volume was obtained with 100 μg in a 0.5 mL centrifuge tube. Freeze-dried and then dissolved in 20 μl of reducing buffer (0.121 g Tris HCl, 7.4 mg EDTA, 5.73 g 胍HCl, dissolved in 9 ml with MilliQ water, adjusted to pH 8.0 with HCL 6N and complete to 10 mL with MilliQ water) . Under 30 s under nitrogen flow, 0.8 μL of 80 mg/mL DTT solution was added and 2H was incubated at 37 ° C in a thermomixer under mixing conditions (800 t/min). The reaction was quenched by the addition of 2 μL of acetic acid and 4 μL of n-propanol. Add 73.2 μL of MilliQ water just prior to injection.

Sample analysis was performed on an Alliance HPLC coupled to a mass spectrometer LCT Premier (Micromass Waters). For LCMS analysis, Xbridge column BEH300 C4 (2.1 x 10 mm, Waters) and a flow rate of 0.25 mL/min were used. The HPLC elution solution was water 0.05% trifluoroacetic acid for solvent A and n-propanol/water/formic acid/trifluoroacetic acid (90/10/0.1/0.02) for solvent B. The flow rate was 0.25 mL/min and the oven temperature was 60 °C. The gradient used for elution is 10 min 95% solvent A to 80% and the slope is 1.5%/min, 30 min at 20% solvent B to 50% and the slope is 1%/min, and 5 min at 80% solvent B The starting condition is then returned to 95% solvent A. Mass spectrometry was performed on top of LCT Premier (Micromass Waters) with a 3,400 V capillary, desolvation temperature of 350 ° C, source temperature of 150 V and a sample cone of 120 V.

- Results:

Regarding the heavy chain (HC), the 11A and 11B graphs show the heavy chains of Hz6F4-1 (Fig. 11A) and Hz6F4-2 (Fig. 11B). In both cases, the three major peaks have the mass of the experiment consistent with the theoretical polypeptide sequence, plus the expected glycoforms (G0F, G1F and G2F, respectively). However, the heavy chain of Hz6F4-1 showed an extra peak, which confirmed the higher heterogeneity compared to Hz6F4-2.

In Figures 12A and 12B, the heavy chains of Hz6F4-1 (A) and Hz6F4-2 (B) were digested with PNGase F to remove the N-glycans. In both cases, the peaks produced by mass spectrometry exhibit the expected mass of the respective polypeptide sequence. The remaining minor peaks are consistent with the saccharification site, with an average of 1 or 2 hexoses. Again, Hz6F4-2 (Fig. 12B) shows much higher homogeneity than Hz6F4-1 (Fig. 12A).

Example 11: In vivo activity of Hz6F4-2 for A-431 xenograft tumor models

The A-431 epidermoid carcinoma cell line was obtained from the American Type Culture Collection and routinely cultured in DMEM (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Sigma). The cells were maintained at 37 ° C in a humidified atmosphere of 95% air / 5% CO 2 .

Athymic (Hsd: Athymic Nude- Foxnlnu ) female nude mice (7 weeks old), obtained from Harlan Sprague Dawley, in a light/dark cycle of 12/12 h and in any available sterile rodent food Water supply. All procedures involving the care of animals and the like are licensed by the Institutional Animal Care and Use Committee of the Centre d'Immunologie Pierre Fabre. Mice were injected subcutaneously into the right flank of each mouse to 10 million A-431 cells during the exponential growth phase. After 5 days, healthy animals with similar tumor volumes and homogenous tumor size groups each assigned to 6 animals were carefully selected. Animals were treated with ip treatment at a dose of 2 mg/dose of Hz6F4-2 at D5 and 1 mg/dose per mouse, twice/week until the end of the experiment. The control group was treated with ip with the corresponding volume of PBS. The tumor volume was evaluated twice a week using the following formula: π/6. length, width, height. The results are shown in Figure 13.

Example 12: In vivo activity of Hz6F4-2 for DU145 xenograft tumor model

The DU145 prostate cancer cell line was obtained from the American Type Culture Collection and routinely cultured in DMEM (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Sigma). The cells were maintained at 37 ° C in a humidified atmosphere of 95% air / 5% CO 2 .

Swiss-Nude (Crl: NU(Ico)- Foxnlnu ) female mice (8 weeks old), obtained from Charles River Laboratories France, in a light/dark cycle of 12/12 h with any available sterility Rodent food and water are supplied. All procedures involving the care of animals and the like are licensed by the Institutional Animal Care and Use Committee of the Centre d'Immunologie Pierre Fabre. Mice were injected subcutaneously into the right flank of each mouse to 10 million DU-145 cells during the exponential growth phase. After 5 days, healthy animals with similar tumor volumes and homogenous tumor size groups each assigned to 6 animals were carefully selected. Animals were treated with ip treatment at a dose of 2 mg/dose of Hz6F4-2 at D5 and 1 mg/dose per mouse, twice/week until the end of the experiment. The control group was treated with ip with the corresponding volume of PBS. The tumor volume was evaluated twice a week using the following formula: π/6. Length, width, height. The results are shown in Figure 14.

Example 13: Inhibition of proliferation of A431 cells with Mab Hz6F4-2

The A431 cell line was sown in 2% serum at different concentrations and incubated with 20 μg/ml of either an unrelated antibody or Mab 6F4 and incubation for 4 or 7 days. Cell proliferation was assessed using ATP measurements.

The results represent the percentage inhibition of cell proliferation in the presence of Mab Hz 6F4-2 compared to the control (n=4 +/- SD). *, P <0.05; **, P < 0.01, the two-sided Student's t test.

The ability of Mab Hz6F4-2 to reduce proliferation of A431 cells was assessed after 4 or 7 days of incubation with different A431 cell concentrations.

As illustrated in Figure 15, a significant reduction in cell count was noted at concentrations of 0.75 x 10 ⁴ and 1.5 x 10 ⁴ cells/cm ² . This inhibition was observed until D4 but increased to 11% ± 7 (p = 0.025) and 20% ± 5 after a period of 7 days of incubation with antibodies of 0.75 x 10 ⁴ and 1.5 x 10 ⁴ cells/cm ² , respectively. p=0.0003).

These results show that Mab Hz6F4-2 significantly reduces cell proliferation of A431 cells.

Example 14: Down regulation of JAM-A by Mab Hz6F4-2

A) The JAM-A performance level is assessed by Western blots. The A431 cell line was sown in serum-free medium at different concentrations for 24 h and at 10 μg/ml whether it was an unrelated antibody or Mab Hz6F4-2. GAPDH was used as a loading control (Fig. 16A).

B) JAM-A and GAPDH were quantified by densitometric analysis. The ratio of JAM-A/GAPDH was determined to assess the down-regulation of JAM-A in cells treated with Mab Hz6F4-2. Results represent mean values of 3 independent experiments +/- SD*, P < 0.05.

C) Cell lines were treated with 10 μg/ml subtype control or Mab Hz6F4-2 for 48 h. The expression level of JAM-A on the cell surface was determined by immunofluorescence staining and visualized by confocal microscopy. JAM-A is expressed using antibodies that bind to the second stage of Alexa Fluor 488.

To assess the mechanism of action of the antibody Hz6F4-2, a series of in vitro experiments were performed to begin the study of JAM-A performance on A431 cells followed by treatment with the antibody. For this purpose, various concentrations of the A431 cell line were incubated with 10 μg/ml of either Hz6F4-2 or a subtype control antibody for 24 h. When the cell line was incubated with the Hz6F4-2 Mab relative subtype control, significant downregulation of JAM-A independent of cell concentration was observed. This observation was quantified by density measurement analysis (Fig. 16B). The results expressed as JAM-A/GAPDH ratios showed no matter what test conditions, noting the reduction in JAM-A from 42 to 52%. The downward regulation observed by Western blots was confirmed by confocal analysis (Fig. 16C), which clearly indicated a dramatic loss of JAM-A after Hz6F4-2 treatment.

Example 15: Effect of Mab Hz6F4-2 on adhesion molecule expression

Cell lines were seeded at 3 x 10 ⁴ cells/cm2 and incubated with 10 μg/ml subtype control or Mab Hz6F4-2 for 24 h. The performance levels of several adhesive molecules are evaluated by Western blots. ITG indicates integrin.

As already illustrated in Figures 16A, B and C, Figure 17 shows that Mab Hz6F4-2 induces a significant downward regulation of JAM-A. However, under the conditions of this test, neither the effect on β1-integrin nor the effects on α5-integrin and αv-integrin were observed, and α5-integrin and αv-integrin were known. It will complex with β1-integrin to form a functional fibronectin receptor (J Cell Sci 2006, 119: 3901). As already shown in other studies, in the case of siRNA to reduce JAM-A gene, E-cadherin and β4-integrin levels after treatment with Hz6F4-2 Mab Nothing has been observed in the quasi-facets.

These results indicate that Mab Hz6F4-2 does not induce a major effect on the level of expression of the adhesive molecule.

Example 16: In vivo activity of hz6F4-2 Mab for the A431 xenograft model.

Athymic (Hsd: Athymic Nude- Foxn1 ^nu ) female nude mice (7 weeks old), obtained from Harlan Sprague Dawley, in a light/dark cycle of 12/12 h with any available sterile rodent food Feed with water. All procedures involving the care of animals and the like are licensed by the Institutional Animal Care and Use Committee of the Centre d'Immunologie Pierre Fabre. Mice were injected subcutaneously into the right flank of each mouse to 10.10 ⁶ A-431 cells during the exponential growth phase. At randomization time, healthy animals with similar tumor volumes and homogenous tumor size groups each assigned to 6 animals were carefully selected. Animals were treated with ip with a loading dose and half of the loading dose was used until the end of the experiment. The control group received ip treatment with the corresponding volume of vehicle (previous experiments using in vivo subtype controls showed no difference in tumor growth compared to the comparative animals treated with vehicle alone). The tumor volume was evaluated twice a week using the following formula: π/6 X length X width X height. Statistical analysis at various time points was performed using Mann-Wbitney Rank Sum Test and SigmaStat software.

The possibility of anti-cancer of Hz6F4-2 Mab is exemplified by the use of A431 epidermoid carcinoma as an additional xenograft model. This in vivo assay in addition to the early tumor (62 mm ³ tumor volume mean) was also tested in established tumors (230 mm ³ and 104 mean tumor volume). The anti-system was ip injected at D4, 14 and 25 after cell engraftment, and the treated mice received one weekly injection at a dose of 10 mg/kg. Treatment started with a loading of 20 mg/kg. Significant inhibition of tumor growth in vivo was observed for all test settings followed by treatment with Hz6F4-2 Mab (Fig. 18). At the termination of treatment, each achieved 57, 70, and 72% inhibition in early, intermediate, and established tumors.

These interesting results demonstrate for the first time that JAM-A plays a role in tumor growth control in established tumors in vivo.

<110> Pierre Faber Pharmaceuticals

<120> Novel homogeneity anthropomorphic anti-proliferative antibodies

<130> D29193

<150> EP 10306286.5

<151> 2010-11-23

<160> 14

<170> PatentIn version 3.3

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Figure 1 shows the alignment between mouse 6F4 VH and each of the selected human V-genes and J-genes;

Figure 2 shows the alignment between mouse 6F4 VH and each of the selected human V-genes and J-genes;

Figure 3 illustrates the binding of JAM-AECD by ELISA; the binding activity of chimeric and anthropomorphic antibodies (A: heavy chain; B: light chain) to JAM-A extracellular domain (ECD) is by ELISA Measurements, where an anti-human CK conjugate was used to detect purified antibodies, dose-dependent binding activity was measured on a plastic coated JAM-A ECD at 450 nm;

Figure 4 illustrates the competitive binding of biotinylated murine 6F4 to JAM-A ECD by ELISA; biotinylated via chimeric and anthropomorphic (A: heavy chain; B: light chain) antibodies Competitive binding of murine 6F4 Mab to JAM-A extracellular domain (ECD) was measured by ELISA; dose-dependent replacement of biotinylated 6F4 was monitored over plastic coated JAM-A ECD;

Figure 5 illustrates competitive binding of antibody binding by JAM-A ECD by ELISA: anthropomorphic heavy chain and anthropomorphic light chain; binding activity of chimeric and anthropomorphic Mab to JAM-A extracellular domain (ECD) (A) is measured by ELISA using an anti-human Cκ conjugate; competition with the biotinylated murine 6F4 Mab(B) via JAM-A extracellular domain (ECD) via chimeric and anthropomorphic antibodies Sex binding is measured by ELISA;

Figure 6 shows the IEF gel for Hz6F4-1 vs. Hz6F4-2 (2 batches);

Figures 7A and 7B show capillary isoelectric focusing (cIEF) for Hz6F4-1 (A) and Hz6F4-2 (B);

Figures 8A and 8B show cation exchange chromatography (CEX) for Hz6F4-1 (A) and Hz6F4-2 (B);

Figures 9A and 9B show the results of mass spectrometry for the entire antibody (IgG) of Hz6F4-1 (A) and Hz6F4-2 (B);

Figures 10A and 10B show the results of mass spectrometry of the entire antibody subjected to PNGase F digestion (IgG) for Hz6F4-1 (A) and Hz6F4-2 (B);

Figures 11A and 11B show the results of mass spectrometry for heavy chain (HC) of Hz6F4-1 (A) and Hz6F4-2 (B);

Figures 12A and 12B show the results of mass spectrometry of heavy chain (HC) digested with PNGase F for Hz6F4-1 (A) and Hz6F4-2 (B);

Figure 13 corresponds to the A-431 xenograft tumor model;

Figure 14 corresponds to the DU-145 xenograft tumor model;

Figure 15 illustrates the inhibition of proliferation of A431 cells with Mab Hz6F4-2;

Figures 16A, B and C illustrate the downward regulation of JAM-A by Mab Hz6F4-2 using A: Western blots, B: densitometry analysis and C: immunofluorescence staining;

Figure 17 illustrates the effect of Mab Hz6F4-2 on the behavior of adhesive molecules by Western blotting;

Figure 18 illustrates the in vivo activity of hz6F4-2 Mab on the A431 xenograft model.

Claims (13)

A method for preparing a homologous variant of a parental humanized antibody or a derivative or antigen-binding fragment thereof, the parentalized humanized antibody comprising a light chain variability field, the light chain variable region having three CDRs sequence identification number 1, 2 and 3, and a heavy chain variable region having three CDRs sequence identification numbers 4, 5 and 6, and the anthropomorphic antibody capable of binding JAM-A, wherein The method comprises the steps of: a) replacing a light chain variable domain of the parentalized humanized antibody or a heavy chain of the parentalized humanized antibody with a neutral amino acid residue or a basic amino acid residue; At least one amino acid residue of a heavy chain variable; and b) selecting whether the substitution results in an increase in the pI of the variant anthropomorphic antibody compared to the parentalized humanized antibody The variant of the anthropomorphic antibody obtained in a) is a highly homogenous variant thereof. A method for producing a homologous variant of a parentalized humanized antibody according to claim 1, wherein in step a): - selected from D1 having the sequence of the light chain variable having sequence number identification number 9. At least one amino acid residue of Q3, Q24 and E55 is substituted with a neutral amino acid residue or a basic amino acid residue, and - is selected from the group having sequence number identification number 10 At least one amino acid residue of E1, Q38, G44, E62 and D77 in the field of chain variability is substituted with a neutral amino acid residue or a basic amino acid residue. A method for producing a homologous variant of a parentalized humanized antibody according to claim 1 or 2, wherein in step a): - selected from the group of light chain variants having sequence number identification number 9 At least one amino acid residue of D1, Q3, Q24 and E55 is substituted with amino acid residues A, R, K and Q, respectively; and - is selected from the heavy chain having sequence number identification number 10) At least one amino acid residue of E1, Q38, G44, E62 and D77 in the variant domain is substituted with amino acid residues Q, R, R, Q and S, respectively. A variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to claim 3, characterized in that it comprises: i) a light chain variability field comprising sequence identification number 11, and ii A heavy chain variability field comprising sequence sequence identification number 12. A variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to claim 4, characterized in that the pI is comprised between 7 and 9, preferably between 7 and 8. . A variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to claim 4 or 5, characterized in that it has a JA of the JAM-A protein of between about 100 nM and about 100 pM. _D, more preferentially between K _D JAM-a protein of between 50 nM and 1 nM. An isolated nucleic acid, characterized in that it is selected from the group consisting of: a) a nucleic acid, DNA or RNA, which encodes an antibody according to any one of claims 4 to 6, or a derivative or antigen-binding fragment; b) a nucleic acid comprising a heavy chain of nucleic acid sequence sequence number 13 and a nucleic acid sequence identification number 14; c) a nucleic acid complementary to a) or b) A nucleic acid as defined. A variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to any one of claims 4 to 7, for use as a medicament. A composition comprising, as one of the active ingredients, a compound which is a variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to any one of claims 4 to 8. A composition as in claim 9 of the patent application for use as a medicament. A variant anthropomorphic antibody, or a derivative or antigen-binding fragment thereof, according to any one of claims 4 to 8, and/or a composition according to any one of claims 9 or 10, For use in the preparation of a medicament for the prevention or treatment of a disease associated with tumor cell proliferation. The use of the scope of claim 11 for the preparation of a medicament for the prevention or treatment of cancer. The use of the scope of claim 12 is characterized in that the cancer is a cancer selected between: prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, multiple myeloma, ovarian cancer, Pancreatic cancer and colon cancer.