Disclosure of Invention
In a first embodiment, the invention relates to an antigen binding protein, or an antigen binding fragment thereof, that is i) specifically binds to the human protein Axl, and ii) is internalized upon binding to said human protein Axl.
More generally, the present invention relates to the use of the protein Axl for the selection of an antigen binding protein, or an antigen binding fragment thereof, which is capable of being internalized upon binding to said target Axl. More specifically, the target is the extracellular domain of Axl.
In this particular aspect, the present invention therefore relates to an in vitro method for screening a compound, or a binding fragment thereof, capable of delivering or internalizing a molecule of interest into a mammalian cell, said molecule of interest being covalently linked to said compound, wherein said method comprises the steps of:
a) selecting a compound capable of specifically binding to the Axl protein, or its extracellular domain (ECD), or an epitope thereof;
b) optionally, covalently linking the molecule of interest or a control molecule to the compound selected in step a) to form a complex;
c) contacting said compound selected in step a) or said complex obtained in step b) with a mammalian cell, preferably a living cell, expressing on its surface the Axl protein or a functional fragment thereof;
d) determining whether the compound or the molecule of interest or the complex has been delivered intracellularly or internalized into the mammalian cell; and
e) the compound is selected as a compound capable of delivering or internalizing a molecule of interest into a living mammalian cell.
In a preferred embodiment, the compound capable of delivering or internalizing a molecule of interest into a living mammalian cell is a protein (also referred to herein as a polypeptide or peptide) or a protein-like compound containing a peptide structure, particularly an amino acid sequence of at least 5, 10, 15 or more amino acid residues, which can be glycosylated.
When the compound capable of delivering or internalizing a molecule of interest into a living mammalian cell is a protein or protein-like compound, the compound is also referred to herein as an "antigen binding protein," which antigen binding protein, or binding fragment thereof, can:
-i) a specific binding protein Axl, preferably a human Axl protein, and
-ii) when said Axl protein is expressed on the surface of said mammalian cell, binding of said protein Axl is followed by internalization into a mammalian cell.
In a preferred embodiment, the living mammalian cell is a human cell, preferably a cell that naturally expresses an Axl protein receptor.
In a particular embodiment, the living mammalian cells in step c) are mammalian cells expressing the recombinant Axl protein on their surface.
In yet another preferred embodiment, the molecule of interest is a cytotoxic molecule (also referred to herein as a cytotoxic or cytostatic agent).
In yet another preferred embodiment, said molecule of interest is covalently linked to said compound capable of binding to the Axl protein using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, more preferably a linker cleavable by a native intracellular compound comprised in a mammalian cell, in particular in the cytoplasm of said mammalian cell.
In yet another preferred embodiment, the compound capable of binding to the Axl protein is an antibody, or a functional binding fragment thereof, specific for the Axl protein, or for an epitope thereof located in the Axl EDC domain.
The selection step of e) can be achieved by any method known to the person skilled in the art for assessing intracellular delivery or internalization. Assays or tests capable of demonstrating or assessing the presence, absence or activity of said compound capable of specifically binding to the Axl protein, or of said complex formed by said compound and said molecule of interest, or of said molecule of interest covalently linked to said compound, are well known to the skilled person (see, hereinafter disclosed some examples of such assays or assays, these tests are not limited to the examples of these tests below).
More specifically, these tests or analyses can be accomplished by FACS, immunofluorescence, flow cytometry, Western blot, cytotoxicity/cytostatic evaluation, and the like.
In this respect, the invention also relates to an in vitro method for preparing a cytotoxic or cytostatic complex capable of delivering a cytotoxic compound into a mammalian cell, preferably a living cell, comprising the steps of:
-covalently linking a cytotoxic agent to a compound:
-i) a compound that specifically binds to the protein Axl, preferably the human Axl protein, and
-ii) when said Axl protein is expressed on the surface of said mammalian cell, said compound is internalized into the mammalian cell after binding to said protein Axl.
Preferably, the compound is a protein-like protein, more preferably an antibody specific for the Axl protein, or for an epitope thereof located in the Axl EDC domain, or a functional binding fragment of said antibody.
In a preferred embodiment, the cytotoxic agent is covalently linked to the anti-Ax 1 antibody or functional fragment thereof using a linker, more preferably a peptide linker, more preferably a cleavable peptide linker, more preferably a linker that can be cleaved by a native intracellular compound as a non-limiting example.
Like other members of the TAM family, the Axl extracellular domain (ECD) has a structure that is close to those of cell adhesion molecules. The Axl ECD is characterized by a combination of two immunoglobulin-like domains followed by two adjacent fibronectin type III-like domains (O' Bryan J.P. et al, mol.cell Biol. (1991).11, 5016-. Two N-terminal immunoglobulin-like domains are sufficient to bind Gas6 ligand (Sasaki T et al, EMBO J. (2006).25, 80-87).
The ECD of the human protein Axl is a 451 amino acid fragment corresponding to amino acids 1-451 of sequence SEQ ID No.29, the sequence of which is shown in the sequence Listing as SEQ ID No. 31. Amino acids 1 to 25 correspond to the signal peptide, and the ECD of the human protein Axl without signal peptide corresponds to amino acids 26 to 451 of sequence SEQ ID No.29, represented by sequence SEQ ID No. 32.
Different modes of internalization have been determined so far. They are directed to proteins or protein complexes that are internalized in the cell. Following endocytosis, most of the membrane proteins or lipids return to the cell surface (recirculation), but some membrane components are delivered to late endosomes (late endosomes) or golgi (Maxfield F.R. & McGraw, t.e. nat. rev. mol. cell biol. (2004).5, 121-.
In a preferred embodiment, the invention relates to an antigen binding protein or antigen binding fragment thereof, which i) specifically binds to the human protein Axl, and ii) is internalized upon binding to said human protein Axl, said antigen binding protein comprising at least an amino acid sequence selected from SEQ ID No.1 to 14, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.1 to 14.
In a most preferred embodiment, the present invention relates to an antigen binding protein or antigen binding fragment thereof, which
i) Specifically binding to the human protein Axl, preferably having the sequence SEQ ID NO.29 or 30 or a naturally occurring variant sequence thereof, and
ii) is internalized upon binding to the human protein Axl,
the antigen binding protein comprises at least an amino acid sequence selected from the group consisting of SEQ ID No.1 to 14.
"binding protein" or "antigen binding protein" refers to a peptide chain having specific or general affinity for another protein or molecule (often referred to as an antigen). When binding is possible, the proteins are allowed to contact and form a complex. The antigen binding protein of the present invention may preferably, but not limited to, an antibody; fragments or derivatives of antibodies, proteins or peptides.
An "antigen-binding fragment" of an antigen-binding protein according to the invention is intended to mean a fragment which retains the ability of the antigen-binding protein to specifically bind to a target (also commonly referred to as an antigen) and which comprises at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least one amino acid residue, at least, Or at least 250 consecutive amino acid residues.
In a preferred embodiment, wherein the antigen binding protein is an antibody, such "antigen binding fragment" is selected from the group consisting of Fv, scFv (sc for single chain), Fab, (Fab')2Fab ', scFv-Fc fragment or diabody, or by chemical modification, e.g.by addition of a poly (alkylene) glycol such as polyethylene glycol ("PEGylation") (the PEGylated fragment is known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab')2-PEG or Fab' -PEG) ("PEG" means polyethylene glycol), or any fragment thereof that enhances its half-life by incorporation into liposomes, said fragment having at least one characteristic CDR of an antibody according to the invention. Preferably, the "antigen-binding fragment" will consist of or comprise a partial sequence of the variable heavy or light chain of the antibody from which it is derived sufficient to retain the same binding specificity as the antibody from which it is derived and sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10, of the affinity of the antibody from which it is derived for the target. Such functional fragments contain at least 5 amino acids, preferably 10, 15, 25, 50 or 100 consecutive amino acids of the antibody sequence from which they are derived.
The term "epitope" refers to a region of an antigen that is bound by an antigen binding protein, including an antibody. An epitope can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant, which is a chemically active molecular surface group, such as an amino acid, sugar side chain, phosphoryl, or sulfonyl group, and in certain embodiments, may have a particular three-dimensional structural characteristic, and/or a particular charge characteristic.
In the present application, the epitope is localized in the extracellular domain of the human protein Axl.
According to a preferred embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, specifically binds to an epitope localized in the extracellular domain of the human protein Axl, preferably having the sequence SEQ ID No.31 or 32 or a native variant sequence thereof.
"specific binding," "specific binding," and the like, are intended to mean that the antigen-binding protein, or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding may be characterized by at least about 1.10-6M or less. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For the avoidance of doubt, this does not mean that the antigen-binding fragment is unable to bind to or interfere with another antigen at low levels. In any event, as a preferred embodiment, the antigen binding fragment binds only to the antigen.
In this sense, "EC50"means an effective concentration of 50%. More precisely, the term half maximal Effective Concentration (EC)50) Drug, antibody or poison concentrations corresponding to half the response between baseline and maximum after a specified exposure time are induced. It is commonly used as a measure of drug efficacy. Thus, EC of the fractionated dose response curve50Represents the concentration of the compound at which 50% of its maximal effect is observed. Quantum dose reversalEC for curve50Representing the concentration of compound at which 50% of the population exhibited a response after a particular period of exposure. Concentration measurements generally follow a sigmoidal curve, increasing rapidly in relatively small concentration changes. This can be determined mathematically by derivation of a best fit line.
As a preferred embodiment, the EC determined in the present invention50The efficacy of the antibodies in binding to Axl ECD exposed on human tumor cells was characterized. Determination of EC Using FACS analysis50And (4) parameters. EC (EC)50The parameters reflect the concentration of antibody that achieves 50% of the maximum binding to human Axl expressed on human tumor cells. Using a four parameter regression curve fitting program (Prism software), each EC50Values were calculated as the midpoint of the dose-response curve. The parameter has been selected to be representative of the physiological/pathological condition.
In one embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, is present in an amount of at least 10-9M, preferably 10-9And 10-12EC between M50Binds to its epitope.
Another embodiment of the invention is a process or method for selecting an antigen binding protein or antigen binding fragment thereof capable of being internalized intracellularly into a mammalian cell, preferably a human cell, preferably a living cell, comprising the steps of:
-i) selecting an antigen binding protein that specifically binds to an Axl protein, preferably its ECD domain or epitope; and
-ii) selecting said antigen binding protein from the previous step i), wherein said antigen binding protein is internalized into a mammalian cell following binding to Axl protein expressed on the surface of said mammalian cell.
In a particular embodiment, the mammalian cell naturally expresses the Axl protein receptor on its surface, or is a mammalian cell, preferably a human cell, expressing a recombinant Axl protein on its surface.
Such a methodThe method or process may include the steps of: i) is selected to be at least 10-9EC of M50An antigen binding protein that specifically binds Axl, and ii) selecting from previous steps an antigen binding protein that is internalized upon binding Axl. The selection step of ii) may be effected by any method known to the person skilled in the art for evaluating internalization. More specifically, the test can be carried out by FACS, immunofluorescence, flow cytometer, western blot, cytotoxicity evaluation, and the like.
Another feature of the antigen binding protein according to the invention is that it does not have any significant activity on the proliferation of tumor cells. More specifically, as shown in the examples below, the antigen binding proteins according to the invention do not have any significant in vitro activity on the proliferative SN12C model.
In oncology, there are a number of mechanisms by which mabs may exert therapeutic effects, but generally are not sufficiently active to produce a lasting benefit. Therefore, several strategies have been used to enhance their activity, particularly by combining them with drugs as chemotherapeutic agents. As an effective alternative to combination protocols, immunotoxins have become a new therapeutic option for the treatment of cancer (Beck a, et al discov.med. (2010), 10, 329-938; Alley s.c., et al j.pharmacol.exp.ther. (2009), 330, 932-938). Antibody Drug Conjugates (ADCs) represent one such approach in which the activity of mabs and drugs can be significantly enhanced by exploiting mAb specificity and the ability to target cytotoxic agents to tumors. Ideally, a mAb will specifically bind to an antigen that is abundantly expressed on tumor cells but is limited in expression on normal cells.
The present invention concerns specific anti-Ax 1 binding proteins, and more specifically specific anti-Axl antibodies, which exhibit a high capacity to be internalized following Axl binding. Such antigen binding proteins are of interest as one of the components of an immuno-drug conjugate, so it carries the attached cytotoxin into the targeted cancer cell. Once internalized, the cytotoxin triggers tumor cell death.
An important key to the success of immunoconjugate therapy is believed to be the specificity of the target antigen and the internalization of the antigen binding protein complex into the cancer cells. Clearly, non-internalized antigens are not able to efficiently deliver cytotoxic agents compared to internalized antigens. The process of internalization is variable from antigen to antigen and depending on a variety of parameters affected by the binding protein. Cell-surface RTKs constitute a family of antigens that are interesting for studying this process.
Among biomolecules, cytotoxins bring about cytotoxic activity, antigen binding proteins used bring about specificity for cancer cells, and carriers are used to enter cells to correctly localize cytotoxins.
Thus, to improve the immunoconjugate molecule, the carrier binding protein must exhibit a high capacity for internalization into the target cancer cell. The efficiency of binding protein-mediated internalization varies significantly depending on the epitope targeted. The selection of potent internalizing anti-Axl binding proteins requires the study of not only Axl down-regulation but also various experimental data after the anti-Axl binding protein enters the cell.
In a preferred embodiment, the internalization of the antigen binding proteins according to the invention can be assessed, preferably by immunofluorescence (as exemplified below in the present application) or by any method or process specific for the internalization mechanism known to those skilled in the art.
In another preferred embodiment, a decrease in the amount of Axl on the cell surface is induced as a result of the complex Axl-antigen binding protein according to the invention being internalized after binding of the binding protein of the invention to the ECD of said Axl. This reduction can be quantified by any method known to those skilled in the art (western blot, FACS, immunofluorescence, etc.).
In embodiments of the invention, this reduction, which thus embodies internalization, is preferably measurable by FACS and expressed as the difference, or Δ, between the Mean Fluorescence Intensity (MFI) measured on untreated cells and the MFI of cells treated with the antigen binding protein according to the invention.
As a non-limiting example of the invention, this delta was determined based on the MFI obtained for untreated cells and for cells treated with the antigen-binding protein of the invention, as described in example 9, using i) human renal tumor SN12C cells after 24 hours incubation with the antigen-binding protein of the invention, and ii) a second antibody labeled with Alexa 488. This parameter is defined as being calculated using the following formula:
since MFI is directly proportional to Axl expressed on the cell surface, this difference between MFI reflects the down-regulation of Axl.
In a more preferred and advantageous aspect, the antigen binding protein or antigen binding fragment thereof of the invention consists of a monoclonal antibody, preferably an isolated Mab, which triggers a delta (MFI) of at least 200, preferably at least 30024hUntreated cells-MFI24hTreated cells).
The antigen binding protein, or antigen binding fragment thereof, according to the invention induces a reduction in MFI of at least 200.
In more detail, the above Δ can be measured according to the following method, which must be considered as an illustrative and non-limiting example:
a) treating and incubating a tumor cell of interest with an antigen binding protein of the invention;
b) parallel treatment of the cells treated in step a) as well as untreated cells with the antigen binding protein of the invention,
c) measuring the MFI (representing the amount of Axl present on the surface) of the treated and untreated cells with a second labeled antibody capable of binding the antigen binding protein, and
d) delta was calculated as the difference of the MFI obtained for untreated cells minus the MFI obtained for treated cells.
In the broadest sense, the terms "antibody" or "immunoglobulin" are used interchangeably and include monoclonal antibodies, preferably isolated mabs (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, or multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity).
More specifically, such molecules consist of glycoproteins comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first complement of the classical complement system (Clq).
Antibodies in the sense of the present invention also include certain antibody fragments thereof. The antibody fragments exhibit the desired binding specificity and affinity regardless of their source or immunoglobulin class (i.e., IgG, IgE, IgM, IgA, etc.), that is, they are capable of specifically binding to the Axl protein with an affinity comparable to that of the full-length antibodies of the invention.
In general, for the preparation of monoclonal Antibodies or functional fragments thereof, in particular of murine origin, it is possible to use the techniques described in particular in the handbook "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726,1988) or the techniques described by Kohler and Milstein for the preparation of hybridomas (Nature,256: 495-.
The term "monoclonal antibody" or "Mab" as used herein refers to an antibody molecule directed against a particular antigen and produced by a monoclonal antibody of a B cell or hybridoma. Monoclonal antibodies may also be recombinant, i.e., produced by protein engineering. In addition, unlike polyclonal antibody preparations which typically include various antibodies directed against different determinants or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The present invention relates to an antibody isolated or obtained from natural sources by purification, or obtained by genetic recombination or chemical synthesis.
A preferred embodiment of the invention is an antigen binding protein, or antigen binding fragment thereof, comprising or consisting of an antibody comprising three light chain CDRs and three heavy chain CDRs; the three light chain CDRs comprise sequences SEQ ID nos. 1,2 and 3, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 1,2 and 3; the three heavy chain CDRs comprise the sequences SEQ ID No.4, 5 and 6, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.4, 5 and 6.
In a more preferred embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, consists of an antibody comprising three light chain CDRs comprising sequences SEQ ID nos. 1,2 and 3; and three heavy chain CDRs comprising sequences SEQ ID No.4, 5 and 6.
In a preferred aspect, the CDR regions or CDRs are intended to represent hypervariable regions of immunoglobulin heavy and light chains as defined by IMGT. Without any conflicting description, CDRs are defined in the present specification according to the IMGT numbering system.
The unique numbering of IMGT, regardless of The antigen receptor, chain type or species, is defined as The comparative variable domain (Lefranc m. -p., Immunology today18,509(1997)/Lefranc m. -p., The Immunologist,7, 132-. In the unique numbering of IMGT, conserved amino acids always have the same position, e.g., cysteine 23 (CYS 1), tryptophan 41 (conserved TRP), hydrophobic amino acid 89, cysteine 104 (CYS 2), phenylalanine or tryptophan 118(J-PHE or J-TRP). The unique numbering of IMGT provides the framework regions (FR 1-IMGT: positions 1 to 26, FR 2-IMGT: 39 to 55, FR 3-IMGT: 66 to 104 and FR 4-IMGT: 118 to 128) and the complementarity determining region CDR 1-IMGT: 27 to 38, CDR 2-IMGT: 56 to 65 and CDR 3-IMGT: 105 to 117. Since the gaps represent unoccupied sites, the CDR-IMGT length (shown between brackets and separated by dots, e.g., [8.8.13]) becomes critical information. The unique numbering of IMGT is used in the 2D scheme and designated IMGT colloids de Perles (Ruiz, M. and Lefranc, M. -P., Immunogenetics,53, 857-.
It is to be understood, however, that, without contradiction in this specification, complementarity determining regions or CDRs mean the hypervariable regions of immunoglobulin heavy and light chains defined by the IMGT numbering system.
In any case, CDRs may also be defined according to the Kabat numbering system (Kabat et al, Sequences of proteins of immunological interest, 5 th edition, U.S. department of Health and Human Services, NIH,1991 and later). There are three heavy chain CDRs and three light chain CDRs. Here, the term "CDR" is used to refer to one or more, or even all, regions comprising the majority of amino acid residues responsible for the antibody binding affinity of the antigen or epitope it recognizes, depending on the circumstances.
The present invention relates to an antigen binding protein or an antigen binding fragment thereof consisting of an antibody comprising three light chain CDRs defined according to the Kabat numbering system and three heavy chain CDRs defined according to the Kabat numbering system, according to the Kabat numbering system; the three light chain CDRs comprise the sequences SEQ ID No.9, 10 and 11, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.9, 10 and 11; the three heavy chain CDRs comprise the sequences SEQ ID nos. 12, 13 and 14, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 12, 13 and 14.
In the meaning of the present invention, "percent identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, which is obtained after optimal alignment, this percentage being purely statistical, the differences between the two sequences being randomly distributed along their length. Comparison of two nucleic acid or amino acid sequences is traditionally performed by comparing the sequences after optimal alignment thereof, which comparison can be performed by segments or by using "alignment windows". In addition to manual comparisons, optimal alignments of the sequences to be compared can be made by the local homology algorithm by Smith and Waterman (1981) (Ad. App. Math.2: 482), by the local homology algorithm by Neddleman and Wunsch (1970) (J.mol. biol.48: 443), by the similarity search by Pearson and Lipman (1988) (Proc. Natl. Acad. Sci. USA 85: 2444) or by computer software using these algorithms (GAP, BESTFIT, TA and TFASTA in the Wisconsin genetics software package, genetics calculation group, 575Science Dr., Madison, Wis, or by the comparison software BLAST NR or FAS P).
The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally aligned sequences, where the nucleic acid or amino acid sequences to be compared may be added or deleted compared to the reference sequence used for optimal alignment between the two sequences. Percent identity is calculated by determining the number of positions of identical amino acid nucleotides or residues between two sequences (preferably between two complete sequences), dividing the number of positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percent identity between the two sequences.
For example, the BLAST program available on the web page http:// www.ncbi.nlm.nih.gov/gorf/bl2.html, "BLAST 2 sequence" (Tatusova et al, "BLAST 2sequences-a new tool for organizing protein and nucleotide sequences", FEMS Microbiol., 1999, Lett.174: 247 250), using default parameters (in particular the parameters "open gap penalty": 5, and "extended gap penalty": 2; the selected matrix is, for example, the program proposed "BLOSUM 62" matrix) may be used; the percent identity between the two sequences to be compared is calculated directly programmatically.
For amino acid sequences that exhibit at least 80%, preferably 85%, 90%, 95% and 98% identity to a reference amino acid sequence, preferred examples include those comprising the reference sequence, certain modifications (in particular deletions, insertions or substitutions of at least one amino acid, truncations or extensions). In the case where one or more consecutive or non-consecutive amino acids are substituted, it is preferred that the amino acid substituted in the substitution is replaced by an "equivalent" amino acid. The expression "equivalent amino acid" is intended here to mean any amino acid which makes it possible to substitute one structural amino acid without altering the biological activity of the corresponding antibody, and any amino acid of those specific examples defined below.
Equivalent amino acids can be determined based on their structural homology to the amino acid they replace, or based on the results of comparative testing of biological activity between the various antigen binding proteins that may be produced.
As a non-limiting example, table 2 below summarizes possible substitutions that may be made without resulting in a significant change in the biological activity of the corresponding modified antigen binding protein; the opposite substitution can naturally be made under the same conditions.
TABLE 2
| Original residues | Substitution |
| Ala(A) | Val,Gly,Pro |
| Arg(R) | Lys,His |
| Asn(N) | Gln |
| Asp(D) | Glu |
| Cys(C) | Ser |
| Gln(Q) | Asn |
| Glu(G) | Asp |
| Gly(G) | Ala |
| His(H) | Arg |
| Ile(I) | Leu |
| Leu(L) | Ile,Val,Met |
| Lys(K) | Arg |
| Met(M) | Leu |
| Phe(F) | Tyr |
| Pro(P) | Ala |
| Ser(S) | Thr,Cys |
| Thr(T) | Ser |
| Trp(W) | Tyr |
| Tyr(Y) | Phe,Trp |
| Val(V) | Leu,Ala |
One embodiment of the present invention relates to an antigen binding protein or antigen binding fragment thereof comprising the light chain variable domain of sequence SEQ ID No.7, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 7; and three heavy chain CDRs comprising the sequences SEQ ID No.4, 5 and 6, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.4, 5 and 6.
According to a preferred embodiment of the invention, the antigen binding protein or antigen binding fragment thereof comprises the light chain variable domain of the sequence SEQ ID No.7, or any sequence exhibiting at least 80% identity with SEQ ID No. 7; and three heavy chain CDRs comprising sequences SEQ ID No.4, 5 and 6.
According to another preferred embodiment of the invention, the antigen binding protein or antigen binding fragment thereof comprises the light chain variable domain of sequence SEQ ID No.7, or any sequence exhibiting at least 80% identity with SEQ ID No. 7.
Another embodiment of the invention relates to an antigen binding protein or antigen binding fragment thereof comprising three light chain CDRs comprising the sequences SEQ ID No.1, 2 and 3, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.1, 2 and 3; and the heavy chain variable domain of sequence SEQ ID No.8, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 8.
According to a preferred embodiment of the invention, the antigen binding protein or antigen binding fragment thereof comprises three light chain CDRs comprising the sequences SEQ ID No.1, 2 and 3; and the heavy chain variable domain of sequence SEQ ID No.8, or any sequence exhibiting at least 80% identity with SEQ ID No. 8.
According to another preferred embodiment of the invention, the antigen binding protein or antigen binding fragment thereof comprises the heavy chain variable domain of sequence SEQ ID No.8, or any sequence showing at least 80% identity with SEQ ID No. 8.
Another embodiment of the invention relates to an antigen binding protein or antigen binding fragment thereof comprising the light chain variable domain of the sequence SEQ ID No.7, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 7; and the heavy chain variable domain of sequence SEQ ID No.8, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 8.
According to a preferred embodiment of the invention, the antigen binding protein or antigen binding fragment thereof comprises the light chain variable domain of the sequence SEQ ID No.7, or any sequence exhibiting at least 80% identity with SEQ ID No.7, and the heavy chain variable domain of the sequence SEQ ID No.8, or any sequence exhibiting at least 80% identity with SEQ ID No. 8.
For greater clarity, table 3a below summarizes the various amino acid sequences corresponding to the antigen binding proteins of the present invention (wherein Mu. ═ murine).
TABLE 3a
A particular aspect of the invention relates to a murine antibody, or a derived compound or antigen binding fragment thereof, characterized in that said antibody further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to murine, in particular human.
Another particular aspect of the invention relates to a chimeric antibody, or a derived compound or antigen-binding fragment thereof, characterized in that said antibody further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to murine, in particular human.
Yet another particular aspect of the invention relates to a humanized antibody, or a derivative compound or antigen-binding fragment thereof, characterized in that the constant regions of the light and heavy chains derived from a human antibody are the lambda or kappa region and the gamma 1, gamma 2 or gamma 4 region, respectively.
Another aspect of the invention is an antigen binding protein consisting of monoclonal antibody 1613F12, said monoclonal antibody 1613F12 derived from hybridoma I-4505, deposited at french institute of pasteur CNCM on day 28, 7, 2011.
According to another aspect, the invention relates to a murine hybridoma capable of secreting an antigen binding protein according to the invention, in particular a murine hybridoma submitted in 2011 at day 7/28 under number I-4505 to the french national culture and collection of microorganisms (CNCM, paris, france). The hybridomas were obtained by fusing Balb/C immunized murine splenocytes/lymphocytes with cells of the myeloma Sp2/O-Ag14 cell line.
According to another aspect, the invention relates to a murine hybridoma capable of secreting an antibody comprising three light chain CDRs comprising the sequences seq id No.1, 2 and 3; and three heavy chain CDRs comprising sequences SEQ ID No.4, 5 and 6, said hybridoma being submitted at 28/7/2011 with accession No. I-4505 to the paris pasteur institute CNCM, france. The hybridomas were obtained by fusing Balb/C immunized murine splenocytes/lymphocytes with cells of the myeloma Sp2/O-Ag14 cell line.
One subject of the present invention is murine hybridoma I-4505, which was deposited at the French Pasteur institute CNCM on 7/28.2011.
Antigen binding proteins of the invention also include chimeric or humanized antibodies.
A chimeric antibody is an antibody that comprises the natural variable regions (light and heavy chains) of an antibody from a given species in combination with the constant regions of the light and heavy chains of an antibody of a species heterologous to the given species.
Antibodies or chimeric fragments thereof can be prepared by using recombinant genetic techniques. For example, chimeric antibodies can be produced by cloning recombinant DNA containing a promoter and sequences encoding the variable region of a non-human (particularly murine) monoclonal antibody of the invention, as well as sequences encoding the constant region of a human antibody. The chimeric antibody according to the invention encoded by one such recombinant gene may be, for example, a murine-human chimera, the variable region from the murine DNA determining the specificity of this antibody, and the constant region from the human DNA determining its isotype. Reference is made to Verhoeyn et al (BioEssays, 8: 74, 1988) for a method for the preparation of chimeric antibodies.
In another aspect, the invention features a binding protein comprised of a chimeric antibody.
In a particularly preferred embodiment, the chimeric antibody or antigen-binding fragment thereof of the invention comprises a light chain variable domain sequence comprising the amino acid sequence SEQ ID No.7 and a heavy chain variable domain sequence comprising the amino acid sequence SEQ ID No. 8.
In another aspect, the invention features a binding protein consisting of a humanized antibody.
By "humanized antibody" is meant an antibody that contains CDR regions from an antibody of non-human origin, the remainder of the antibody molecule being derived from one (or several) human antibodies. In addition, certain framework segment residues (referred to as FR) can be modified to retain binding affinity (Jones et al, Nature, 321: 522-525, 1986; Verhoeyen et al, Science, 239: 1534-1536, 1988; Riechmann et al, Nature, 332: 323-327, 1988).
Humanized antibodies or fragments thereof of the invention can be prepared by techniques known to those skilled in the art (such as, for example, those described in Singer et al, J.Immun., 150: 2844-2857, 1992; Mountain et al, Biotechnology. Genet. Eng. Rev., 10: 1-142, 1992; and Bebbington et al, Bio/Technology, 10: 169-175, 1992). Preferably such humanized antibodies are used in methods relating to in vitro diagnosis or in vivo prophylactic and/or therapeutic treatment. Other humanization techniques known to the person skilled in the art are also known such as, for example, the "CDR grafting" technique described in PDL in patents EP0451216, EP0682040, EP0939127, EP0566647 or US5,530,101, US6,180,370, US5,585,089 and US5,693,761. U.S. Pat. Nos. 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.
In addition, the invention also relates to humanized antibodies produced by the murine antibodies described above.
In a preferred manner, the light and heavy chain constant regions from a human antibody are the lambda or kappa and gamma 1, gamma 2 or gamma 4 regions, respectively.
In a preferred embodiment, the present invention relates to an antigen binding protein consisting of a humanized antibody, or an antigen binding fragment thereof, comprising a light chain variable domain comprising the sequence SEQ ID No.36, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 36; and three heavy chain CDRs comprising sequences SEQ ID No.4, 5 and 6.
Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a light chain variable domain of a sequence selected from SEQ ID No.37-47, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 37-47; and three heavy chain CDRs comprising sequences SEQ ID No.4, 5 and 6.
"any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.36 or 37-47" is intended to mean a sequence presenting the three light chain CDRs SEQ ID No.1, 2 and 3 and, furthermore, exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID No.36 or 37-47 outside the corresponding CDR sequences (i.e. SEQ ID No.1, 2 and 3).
For clarity, table 3b below summarizes the various amino acid sequences corresponding to the light chain (VL) of the humanized antigen binding protein of the present invention (wherein Hz. is humanized).
TABLE 3b
In one embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, comprises a light chain variable domain selected from the group consisting of:
i) a light chain variable domain of the sequence SEQ ID NO.7, or any sequence exhibiting at least 80% identity with SEQ ID NO.7,
ii) the light chain variable domain of the sequence SEQ ID No.36, or any sequence exhibiting at least 80% identity with SEQ ID No. 36; and
iii) the light chain variable domain of the sequence SEQ ID No.37-47, or any sequence exhibiting at least 80% identity with SEQ ID No. 37-47.
In a preferred embodiment, the present invention relates to an antigen binding protein consisting of a humanized antibody, or an antigen binding fragment thereof, comprising a heavy chain variable domain comprising the sequence SEQ ID No.48, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 48; and three light chain CDRs comprising sequences SEQ ID No.1, 2 and 3.
Another embodiment of the invention relates to an antigen binding protein, or an antigen binding fragment thereof, comprising a heavy chain variable domain selected from the sequence of SEQ ID nos. 49-68, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID nos. 49-68; and three light chain CDRs comprising sequences SEQ ID No.1, 2 and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No.48 and 49-68" is intended a sequence presenting the three heavy chain CDRs SEQ ID No.4, 5 and 6 and, furthermore, exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with the full sequence SEQ ID No.48 and 49-68 outside the corresponding CDR sequences, i.e. SEQ ID No.4, 5 and 6.
For clarity, table 3c below summarizes various amino acid sequences corresponding to the heavy chain (VH) of the humanized antigen binding protein of the present invention (wherein Hz. is humanized).
TABLE 3c
In one embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, comprises a heavy chain variable domain selected from the group consisting of:
i) a heavy chain variable domain of sequence SEQ ID No.8, or any sequence exhibiting at least 80% identity with SEQ ID No. 8;
ii) the heavy chain variable domain of sequence SEQ ID No.48, or any sequence exhibiting at least 80% identity with SEQ ID No. 48; and
iii) a heavy chain variable domain of the sequence SEQ ID No.49-68, or any sequence exhibiting at least 80% identity with SEQ ID No. 49-68.
In one embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, comprises the light chain variable domain of sequence SEQ ID No.36, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 36; and the heavy chain variable domain of sequence SEQ ID No.48, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 48.
In another embodiment of the invention, the antigen binding protein, or antigen binding fragment thereof, comprises a light chain variable domain of a sequence selected from SEQ ID nos. 37-47, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95%, and 98% identity to SEQ ID nos. 37-47; and a heavy chain variable domain selected from the sequence of SEQ ID No.49-68, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID No. 49-68.
In one embodiment of the invention, an antigen binding protein, or antigen binding fragment thereof, comprises:
i) a light chain variable domain of the sequence SEQ ID No.7, 36 or 37-47, or any sequence exhibiting at least 80% identity with SEQ ID No.7, 36 or 37-47; and
ii) the heavy chain variable domain of sequence SEQ ID No.8, 48 or 49-68, or any sequence exhibiting at least 80% identity with SEQ ID No.8, 48 or 49-68.
A novel aspect of the present invention relates to an isolated nucleic acid characterized in that it is selected from the following nucleic acids (including any degenerate genetic code):
a) a nucleic acid encoding an antigen binding protein or antigen binding fragment thereof according to the invention;
b) a nucleic acid, comprising:
-a nucleic acid sequence selected from SEQ ID Nos. 15 to 28 and 69 to 99, or
-a nucleic acid sequence comprising six nucleic acid sequences SEQ ID Nos. 15 to 20, or
-a nucleic acid sequence comprising the two nucleic acid sequences SEQ ID nos. 21, 22 or two nucleic acid sequences wherein one part is selected from SEQ ID nos. 69 to 79 and the other part is selected from SEQ ID nos. 80 to 99;
c) a nucleic acid complementary to the nucleic acid as defined in a) or b); and
d) nucleic acids, preferably nucleic acids having at least 18 nucleotides, which are capable of hybridizing under highly stringent conditions with a nucleic acid sequence as defined in part a) or b) or with a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with a nucleic acid sequence as defined in part a) or b).
Table 4a below summarizes various nucleotide sequences for the binding proteins of the invention (wherein Mu. ═ murine).
TABLE 4a
For clarity, table 4b below summarizes the various nucleotide sequences corresponding to the light chain (VL) of the humanized antigen binding proteins of the present invention (wherein Hz. is humanized).
TABLE 4b
For clarity, table 4c below summarizes various nucleotide sequences corresponding to the heavy chain (VH) of the humanized antigen binding protein of the present invention (wherein Hz. is humanized).
TABLE 4c
The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this specification to denote the precise nucleotide sequence of a defined nucleic acid fragment or region, with or without modification, which is double-stranded DNA, single-stranded DNA or a transcript of said DNA, with or without non-natural nucleotides.
The sequences of the invention have been isolated and/or purified, i.e.they are sampled, directly or indirectly (e.g.in one copy), the environment of which has been at least partially altered. Isolated nucleic acids obtained by recombinant genetics (e.g., in the manner of host cells), or obtained by chemical synthesis, are also to be mentioned herein.
By "a core sequence showing at least 80%, preferably 85%, 90%, 95% and 98% percent identity after optimal alignment with a preferred sequence" is meant that the core sequence shows certain modifications (especially site-directed modifications) relative to the reference sequence, such as in particular deletions, truncations, extensions, chimeric fusions and/or substitutions. Preferably, these sequences encode an amino acid sequence which is identical to the reference sequence (which involves the degeneracy of the genetic code), or a complementary sequence which is capable of specifically hybridizing to the reference sequence, preferably under high stringency conditions, particularly those defined below.
Hybridization under highly stringent conditions means that conditions involving temperature and ionic strength are selected in such a way as to allow for the maintenance of hybridization between two complementary DNA fragments. On a purely illustrative basis, the highly stringent conditions of the hybridization steps described above for the purpose of defining the polynucleotide fragments are advantageously as follows.
DNA-DNA or DNA-RNA hybridization was performed in two steps: (1) prehybridization in phosphate buffer (20mM, pH7.5, containing 5 XSSC (1 XSSC corresponds to a solution of 0.15M NaCl +0.015M sodium citrate), 50% formamide, 7% Sodium Dodecyl Sulfate (SDS), 10 XDenhardt's, 5% dextran sulfate, and 1% salmon sperm DNA) at 42 ℃ for three hours; (2) initial hybridization was performed at a temperature dependent on the length of the probe (i.e., 42 ℃ for probes greater than 100 nucleotides long) for 20 hours, followed by two 20-minute washes in 2 XSSC + 2% SDS at 20 ℃ and one 20-minute wash in 0.1 XSSC + 0.1% SDS at 20 ℃. For probes greater than 100 nucleotides in length, a final wash is performed at 60 ℃ in 0.1 XSSC + 0.1% SDS for 30 minutes. For longer or shorter oligonucleotides, one skilled in the art can adjust the highly stringent hybridization conditions described above for a polynucleotide of a particular size according to the procedures described in Sambrook et al (Molecular cloning: a Laboratory Manual, Cold spring harbor Laboratory; third edition, 2001).
The invention also relates to vectors comprising the nucleic acids according to the invention.
The invention is particularly directed to cloning and/or expression vectors comprising such nucleotide sequences.
The vectors of the invention preferably comprise elements which allow for the expression and/or secretion of the nucleotide sequence in a given host cell. The vector must therefore contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be able to be maintained in a stable manner in the host cell and optionally have specific signals directing secretion of the translated protein. Depending on the host cell used, the skilled person will be able to select and optimize these various elements. For this purpose, the nucleotide sequence can be inserted into an autonomously replicating vector in the chosen host or an integrating vector for the chosen host.
Such vectors are prepared in a manner commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods such as lipofection, electroporation, heat shock or chemical methods.
The vector is, for example, a plasmid or a virus-derived vector. They are used to transform host cells to clone or express the nucleotide sequences of the present invention.
The invention also includes isolated host cells transformed with or comprising the vectors of the invention.
The host cell can be selected from prokaryotic or eukaryotic systems, such as bacterial cells, for example, but also yeast cells or animal cells, in particular mammalian cells (except human). Insect or plant cells can also be used.
The invention also relates to animals (other than humans) having transformed cells according to the invention.
Another aspect of the invention relates to a method for producing an antigen binding protein, or an antigen binding fragment thereof, according to the invention, characterized in that the method comprises the following steps:
a) culturing a host cell according to the invention in a culture medium and under suitable culture conditions; and
b) recovering the thus produced antigen binding protein, or one of its antigen binding fragments, from the culture medium or from the cultured cells.
The transformed cells according to the invention can be used in a process for the preparation of recombinant antigen-binding proteins according to the invention. A method for preparing a recombinant form of an antigen-binding protein according to the invention is also comprised in the invention, characterized in that said method uses a vector according to the invention and/or a cell transformed with a vector according to the invention. Preferably, the cells transformed with the vector according to the invention are cultured under conditions allowing the expression of the aforementioned antigen binding protein and the recovery of the recombinant protein.
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 which facilitate secretion in such prokaryotic or eukaryotic systems. The vectors according to the invention carrying such sequences can therefore advantageously be used for the production of recombinant proteins to be secreted. Indeed, the fact that the protein is present in the cell culture supernatant rather than in the host cell will facilitate the purification of these recombinant proteins of interest.
The antigen binding proteins of the present invention can also be prepared by chemical synthesis. One such preparation process is also subject of the present invention. The person skilled in the art is 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, second edition pp71-95) or partial Solid phase techniques, by concentrating the fragments or by conventional synthesis in solution. The invention also includes polypeptides obtained by chemical synthesis and capable of comprising the corresponding unnatural amino acid.
The invention also includes antigen binding proteins or antigen binding fragments thereof as may be obtained by the methods of the invention.
According to a particular aspect, the present invention relates to the above-described antigen binding protein, or antigen binding fragment thereof, for use as a targeting product for delivering a cytotoxic agent to a host target site consisting of an epitope localized in the extracellular domain of the protein Axl, preferably the extracellular domain of the human protein Axl, more preferably the extracellular domain of the human protein Axl having the sequence SEQ id No.31 or 21, or a native variant sequence thereof.
In a preferred embodiment, the host target site is a target site of a mammalian cell, more preferably a human cell, more preferably a cell expressing the Axl protein naturally or by means of genetic recombination.
The present invention relates to immunoconjugates comprising an antigen binding protein as described in the specification conjugated to a cytotoxic agent.
In the sense of the present invention, the expression "immunoconjugate" or "immuno-conjugate" generally refers to a compound comprising at least one localization product, wherein the localization product is physically linked to one or more therapeutic agents, thereby producing a highly targeted compound.
In a preferred embodiment, such therapeutic agents consist of cytotoxic agents.
By "cytotoxic agent" or "cytotoxin" is meant an agent that, when administered to a subject, treats or prevents the development of cell proliferation, preferably cancer development in the body of the subject, by inhibiting or preventing cell function and/or causing cell death.
Many cytotoxic agents have been isolated or synthesized and made capable of inhibiting the proliferation of cells, or (if not specified) at least significantly destroying or reducing tumor cells. However, the toxic activity of these agents is not limited to tumor cells, but non-tumor cells are also affected and can be destroyed. More specifically, side effects are observed in rapidly renewing cells, such as hematopoietic or epithelial cells, especially mucous membranes. By way of illustration, by using such cytotoxic agents, cells of the gastrointestinal tract are greatly affected.
It is also an object of the present invention to provide a cytotoxic agent which makes it possible to limit the side effects on normal cells while at the same time retaining a high cytotoxicity on tumor cells.
More specifically, cytotoxic agents may preferably include, but are not limited to, drugs (i.e., "antibody drug conjugates"), toxins (i.e., "immunotoxins" or "antibody toxin conjugates"), radioisotopes (e.g., "radioimmunoconjugates" or "antibody radioisotope conjugates"), and the like.
In a first preferred embodiment of the invention, the immunoconjugate consists of a binding protein linked to at least one drug or pharmaceutical product. When the binding protein is an antibody, or antigen-binding fragment thereof, such an immunoconjugate is referred to as an antibody drug conjugate (or "ADC").
In a first embodiment, such a drug may be described in terms of its mode of action. As non-limiting examples, alkylating agents (such as nitrogen mustards, alkyl sulfonates, nitrosoureas, oxazophorin (no corresponding intermediate), aziridines or ethylenimines), antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, chelating agents, iron absorption stimulators, cyclooxygenase inhibitors, phosphodiesterase inhibitors, DNA synthesis inhibitors, apoptosis stimulators, thymidylate inhibitors, T cell inhibitors, interferon agonists, ribonucleoside triphosphate reductase inhibitors, aromatase inhibitors, estrogen receptor antagonists, tyrosine kinase inhibitors, cell cycle inhibitors, taxanes, tubulin inhibitors, angiogenesis inhibitors, macrophage stimulators, neurokinin receptor antagonists, cannabinoid receptor agonists, angiotensin receptor antagonists, estrogen receptor antagonists, and the like may be mentioned, Dopamine receptor agonists, granulocyte stimulating factor agonists, erythropoietin receptor agonists, somatostatin receptor agonists, LHRH agonists, calcium sensitizers, VEGF receptor antagonists, interleukin receptor antagonists, osteoclast inhibitors, free radical formation stimulators, endothelin receptor antagonists, vinca alkaloids, anti-hormones or immunomodulators or any other new drug meeting the criteria of cytotoxic or toxin activity.
Such drugs are cited, for example, in VIDAL2010 on pages pertaining to compounds attached to the column "cytotoxins" in oncology and hematology, and these cytotoxic compounds cited with reference to this document are cited herein as preferred cytotoxic agents.
More specifically, but not limited to, the following drugs are preferred according to the invention: dichloromethyldiethylamine, chlorambucil (chlorambucol), melphalan, hydrochloride (chloredrate), pipobroman (pipobromen), prednimustine (prednimustin), pyro (disoic) -phosphate, estramustine, cyclophosphamide, altretamine (altretamine), trofosfamide, sulfoifosfamide (sulfoifomide), ifosfamide, thiotepa, triethylamine, altetramine (no corresponding intermediate), carmustine, streptozocin, fotemustine, lomustine, busulfan, troosulfan, improsulfan, dacarbazine, cisplatin, oxaliplatin, lobaplatin (heptaptin), miriplatin (miriplatin) hydrate, carboplatin, methotrexate, meretrix, 5-fluorouracil, 5-desoxyuracil, 6-D-6-D, arabinoside, 6-thiogalactoside (6-D), arabinoside, and other active ingredients, Nelarabine, 6-thioguanine (6-TG), chlorodeoxyadenosine, 5-azacytidine, gemcitabine (gemcitabine), cladribine (cladribine), deoxycoformycin, tegafur, pentostatin, doxorubicin, daunomycin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide, pentoxitin, amrubicin hydrochloride, pirarubicin, etiloamidinium, zorubicin, epirubicin, idarubicin and teniposide, razine, marimastat, batistat, colomastat, CGS-27023A, haloketone, neovaquone-3, neovavastatine, Thalidomide, CDC501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferon-alpha, EMD121974, interleukin-12, IM862, angiogenesis inhibitor, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene (no corresponding intermediate), anastrozole, letrozole, exemestane, flutamide, nilutamide, ambroxone, cyproterone acetate, finasteride, cimetidine, bortezomib, velcade, bicalutamide, cyproterone, flutamide, fulvestrant (fulvestrant), exemestane, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, retinoids, rexinexine (no corresponding intermediate), interleukin (OCT), plain-43), plain-intermediate (corresponding intermediate), norfloxacin, vallisine (no corresponding intermediate), norfloxacin, valliseline (43), norfloxacin, vallisine, norfloxacin, nor, Interleukin-2, tasonamine (tasonermine), lentinan, tezomib, roquinacre, pidotimod, pegase, carmustine (thymopentine), poly I: C. procodazol (no corresponding intermediate), Tic BCG, Corynebacterium parvum, NOV-002, ukrain (no corresponding intermediate), levamisole, 1311-chTNT, H-101, simethin, interferon alpha 2a, interferon alpha 2b, interferon gamma 1a, interleukin-2, muberelin, Rexin-G, tesile, aclacinomycin, actinomycin, arglabin (no corresponding intermediate), asparaginase, carzinophilin (no corresponding intermediate), tryptomycin, daunomycin, folinic acid, maxolol, neocarzinostatin, pellomycin, sarcomycin, solanine, trabectedin, streptozocin, testosterone, intracytocatechin, sinecebits (no corresponding intermediate), alisertiol, belocochloin (no corresponding intermediate), estramustine, acetate, Etoposide, flumethosterone, formestane, fosfetrol (no corresponding intermediate), goserelin acetate, hexylaminolevulinic acid, histrelin, hydroxyprogesterone, ixabepilone, leuprorelin, medroxyprogesterone acetate, megestrol acetate, methylprednisolone, miltefosine, dibromomannitol, nandrolone phenylpropionate, norethindrone acetate, prednisolone, prednisone, temsirolimus, testolactone, triamcinolone, triptorelin, vapreotide acetate, neat stastine ester, amsacrine, arsenic trioxide, bisantrene hydrochloride, chlorambucil, trimethobranchiastyrene (chlorertinisene), cisplatin, cyclophosphamide, diethylstilbestrol, altretamine, hydroxyurea, lenalidomide, lonidamine, mechlorethanamine (no corresponding intermediate), platinum dapsone, nefotemeportan, nemorphine hydrochloride, pamidronate, meprobamate, medetoposide, medroxil, medroxypro, medetoposide, medrox, Porfimer sodium, ramosestine, razoxane, semustine, sobuzosin, methanesulfonate, triethylenemelamine, zoledronic acid, camostat mesylate, fadrozole HCl, nafoxidine, aminoglutethimide, carmofur, clofarabine, cytosine arabinoside, decitabine, doxifluridine, enocitabine, fludarabnephospate (without corresponding intermediate), fluorouracil, tegafur, uracil mustard, abarelix, bexarotene, ratetrexed (without corresponding intermediate), tamibarotene, temozolomide, vorinostat, megestrol, clophosphonate disodium, levamisole, nano iron oxide, iron isoaltoside (without corresponding intermediate), celecoxib, ibudilast, bendamustine, hexamethyl, melamine, dibromodulcitol, sirolimus, prasuzuril, TS-1, trarsoproxil, troxer, troxerutin, trexapride, trematode, clobetamethamine, clobetamethadone, clotrexadine, valtrexadine, clotrexapril, clomiprid, clobetadine, clomipramine, clorac, clomipide, clomip, Degarelix, toremifene citrate, histamine dihydrochloride, DW-166HC, dacridine, decitabine, irinotecan hydrochloride (without corresponding intermediate), amsacrine, romidepsin, tretinoin, cabazitaxel, vandetanib, lenalidomide, ibandronic acid, miltefosine, vitespen (without corresponding intermediate), mivampitide, nadroparin, granisetron, ondansetron, tropisetron, aripride, ramosetron, dolasetron mesylate, fosaprepitant, marijuron, aprepitant, dronabinol-10721, dihydroergometride maleate, eperidol (without corresponding intermediate), defibrotide, bidagliptin ester, filgrastim, pefilgrastim, redutux, reduture's TY', ramucidol, interleukin, Vasimuelel (without corresponding intermediate), vacrex-M, vallisine, leucite, and so, Acetyl L-carnitine, donepezil hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate, icodextrin, leuprorelin, metbylphenidate (no corresponding intermediate), octreotide, amlexanox, plerixafor, menatetrenone, anethole dithienone, anethole, cinamide dithioleithionine (no corresponding intermediate), doxercalciferol, cinacalcet hydrochloride, alfacacet, rolimus, thyroglobulin, thymalfasin, imiquimod, everolimus, sirolimus, H-101, lasofoxifene, trostandin, incadronic acid, ganglioside, modafinil sodium, verteporfin (no corresponding intermediate), minophosphonic acid, zoledronic acid, gallium nitrate, alendronate sodium, etidronate disodium, pamidronate, dutasteride, stiboglutamide, dexrazoxane, azulenide, azulenomethionine, amiodamine, amiodarone, amitriptyline, WF 10-WF, fosamifostine, amifostine, WF-10, Temoporphine, dabipostin alpha, ancystin, sargrastim, palifermin, R-744, nepedmin, opurpreinterleukin, denileukin diftotox (no corresponding intermediate), creitase (crisantacaspase), buserelin, deserelin, lanreotide, octreotide, pilocarpine, bosentan, calicheamicin, maytansine and nicotinyl.
For more details, those skilled in the art can refer to "AssociationDesEngineers de Chimie Th rapeutique "edited and titled" traitede Chimie Th rapeutique, volume 6, Meta details antisense et perspectives dans le traitementes, TEC&DOC edition, 2003 ".
In a second preferred embodiment of the invention, the immunoconjugate consists of a binding protein linked to at least a radioisotope. When the binding protein is an antibody or antigen-binding fragment thereof, such an immunoconjugate is referred to as an antibody radioisotope conjugate (or "ARC").
For selective destruction of tumors, the antibody may comprise a highly radioactive atom. Various radioactive isotopes are useful for producing ARC, such as, but not limited to At211、C13、N15、O17、Fl19、I123、I131、I125、In111、Y90、Re186、Re188、Sm153、tc99m、Bi212、P32、Pb212And radioactive isotopes of Lu, gadolinium, manganese or iron.
Any method or process known to those skilled in the art may be used to incorporate such a radioisotope into an ARC (see, e.g., "Monoclonal Antibodies in Immunoscintigraphy," Chatal, CRC Press 1989). As a non-limiting example, tc99m or I123、Re186、Re188And In111Attachment may be through a cysteine residue. Y is90Attachment may be through lysine residues. I is123IODOGEN method can be used for attachment (Fraker et al (1978) biochem. Biophys. Res. Commun.80: 49-57).
Various examples may be mentioned to illustrate the general knowledge of a person skilled in the art of ARC, for exampleIt is a CD20 resisting sheetCloning antibodies and In bound by thiourea linker chelators111Or Y90Radioisotope-composed ARCs (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7): 766-77; Wiseman et al (2002) Blood99(12): 4336-42; Witzig et al (2002) J. Clin. Oncol.20(10): 2453-63; Witzig et al (2002) J. Clin. Oncol.20(15): 3262-69); orIt consists of an anti-CD 33 antibody linked to calicheamicin (U.S. Pat. No.4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). Recently, mention may also be made of the ADCs known as Adcetris (corresponding to Brentuximab vedotin (without corresponding to the translation)), which have recently been accepted by the FDA for the treatment of Hodgkin's lymphoma (Nature, Vol 476, pp380-381,2011, 8/25).
In a third preferred embodiment of the invention, the immunoconjugate consists of a binding protein linked to a toxin. When the binding protein is an antibody or antigen-binding fragment thereof, such an immunoconjugate is referred to as an antibody toxin conjugate (or "ATC").
Toxins are potent and specific poisons produced by living organisms. They are generally composed of chains of amino acids, which can vary in molecular weight, between a few hundred (peptides) and one hundred thousand (proteins). They may also be low molecular weight organic compounds. Toxins are produced by many organisms, such as bacteria, fungi, algae, and plants. Many of them are very toxic, with toxicity orders of magnitude stronger than that of neurological agents.
Toxins used for ATC may include, but are not limited to, all kinds of toxins that may exhibit their cytotoxic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Canavan A chain, alpha-sarcin, Aleurites fordii protein, dianthin, Phytolaca americana protein (without corresponding intermediate) (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, Jatropha curcin, crotin, sapaonaria officinalis (without corresponding intermediate) inhibitor, gelonin, mitogellin (without corresponding intermediate), restrictocin, phenomycin, enomycin and tricothecene (without corresponding intermediate).
Also contemplated herein are small molecule toxins such as dolastatin, auristatin (no corresponding intermediate), trichothecene, and CC1065, and derivatives of these toxins that have toxin activity. Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have anti-cancer and antifungal activity.
"linker", "linker unit" or "linkage" refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches a binding protein to at least one cytotoxic agent.
Linkers can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methylethyltriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating cytotoxic agents to a localization system. Other cross-linker reagents may be BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., Pierce Biotechnology, Inc. from Rockford,111, U.S.).
The linker may be "non-cleavable" or "cleavable".
In preferred embodiments, it comprises a "cleavable linker" to facilitate release of the cytotoxic agent within the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker may be used. In preferred embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent from the binding protein in the intracellular environment.
For example, in some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., a lysosome or endosome or caveolae (caveolea)). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including, but not limited to, lysosomal or endosomal proteases). Typically, the peptidyl linker is at least 2 amino acids long, or at least 3 amino acids long. Cleavage agents may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in release of the active drug in the target cell. For example, peptidyl linkers (e.g., Phe-Leu or Gly-Phe-Leu-Gly linkers) that can be cleaved by cathepsin B, a thiol-dependent protease that is highly expressed in cancer tissues, can be used. In particular embodiments, the peptidyl linker that can be cleaved by an intracellular protease is a Val-Cit linker or a Phe-Lys linker. One advantage of using intracellular proteolytic release of the cytotoxic agent is that the agent is generally attenuated when conjugated and the serum stability of the conjugate is generally higher.
In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, the linker, which is pH sensitive under acidic conditions, is hydrolysable. For example, acid-labile linkers that can be hydrolyzed in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. Such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH5.5 or 5.0 (approximately the pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to the therapeutic agent, for example, through an acylhydrazone bond).
In still other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including those that can be formed, for example, using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene), SPDB, and SMPT.
As a non-limiting example of a non-cleavable or "non-reducible" linker, the immunoconjugate trastuzumab-DM 1(TDM1) which combines trastuzumab with a chemotherapeutic agent, maytansine, may be mentioned (Cancer Research 2008; 68 (22); November15, 2008).
In a preferred embodiment, the immunoconjugates of the invention can be prepared by any method known to those skilled in the art, such as, but not limited to, i) reacting a nucleophilic group of an antigen binding protein with a bivalent linker reagent and then with a cytotoxic agent or ii) reacting a nucleophilic group of a cytotoxic agent with a bivalent linker reagent and then with a nucleophilic group of an antigen binding protein.
When the antigen binding protein is glycosylated, nucleophilic groups on the antigen binding protein include, but are not limited to, the N-terminal amino group, side chain amino groups such as lysine, side chain thiol groups, and hydroxyl or amino groups of sugars. Amines, thiols, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on linker moieties to form covalent bonds, linker reagents including, but not limited to, active esters such as NHS esters, HOBt esters, haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides; aldehyde, ketone, carboxyl and maleimide groups. Antigen binding proteins may have reducible interchain disulfide bonds, i.e., cysteine bridges. The antigen binding protein may be rendered reactive by treatment with a reducing agent such as DTT (dithiothreitol) to conjugate with a linker reagent. Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced to the antigen binding protein by any reaction known to those skilled in the art. As a non-limiting example, a reactive thiol group may be introduced into an antigen binding protein by introducing one or more cysteine residues.
Immunoconjugates can also be produced by modifying an antigen binding protein to introduce an electrophilic moiety that can react with a nucleophilic substituent of a linker reagent or cytotoxic agent. The saccharide of the glycosylated antigen binding protein may be oxidized to form an aldehyde or ketone group, which may react with the amino group of the linker reagent or cytotoxic agent. The resulting imine Schiff base groups may form stable linkages or may be reduced to form stable amine linkages. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antigen binding protein with galactose oxidase or sodium periodate may produce carbonyl groups (aldehydes and ketones) in the protein, where the carbonyl groups may react with appropriate groups on the drug. In another embodiment, a protein containing an N-terminal serine or threonine residue can be reacted with sodium periodate, resulting in the production of an aldehyde in place of the first amino acid.
In certain preferred embodiments, the linker unit may have the general formula:
--Ta--Ww--Yy--
wherein:
-T-is an extension (stretcher) unit;
a is 0 or 1;
-W-is an amino acid unit;
w is independently an integer from 1 to 12;
-Y-is a spacer unit;
y is 0,1 or 2.
The extension unit (-T-) is present, and the antigen binding protein is linked to the amino acid unit (-W-). Useful functional groups that may be present in the antigen binding protein, whether native or chemically treated, include sulfhydryl, amino, hydroxyl, anomeric hydroxyl of carbohydrates, and carboxyl groups. Suitable functional groups are mercapto and amino groups. Sulfhydryl groups, if present, can be generated by reducing intermolecular disulfide bonds of the antigen binding protein. Alternatively, sulfhydryl groups may be generated by reacting the amino group of a lysine moiety of an antigen binding protein with 2-iminothiolane or other sulfhydryl generating reagent. In particular embodiments, the antigen binding protein is a recombinant antibody and is engineered to carry one or more lysines. More preferably, the antigen binding proteins may be engineered to carry one or more cysteines (see ThioMabs).
In certain embodiments, the extension unit forms a bond with a sulfur atom of the antigen binding protein. The sulfur atom may be derived from a sulfhydryl (-SH) group of a reduced antigen binding protein.
In certain other embodiments, the extension unit is linked to the antigen binding protein via a disulfide bond between the sulfur atom of the antigen binding protein and the sulfur atom of the extension unit.
In other embodiments, the reactive group of the extension comprises a reactive site that can react with an amino group of the antigen binding protein. The amino group may be arginine or lysine. Suitable amine reactive sites include, but are not limited to, active esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, an anhydride, an acid chloride, a sulfonyl chloride, an isocyanate, and an isothiocyanate.
In yet another aspect, the reactive group of the extension comprises a reactive site that can react with a modified carbohydrate group present on the antigen binding protein. In one embodiment, the antigen binding protein is enzymatically glycosylated to provide a carbohydrate moiety (note that when the antigen binding protein is an antibody, the antibody is typically naturally glycosylated). The carbohydrate may be lightly oxidized with a reagent such as sodium periodate to give an oxidized carbohydrate whose carbonyl units may be condensed with an extension containing a functional group such as hydrazine, oxime, reactive amine, hydrazine, thiosemicarbazide, hydrazine carboxylate, or aryl hydrazide.
The amino acid unit (-W-) connects the extender unit (-T-) to the spacer unit (-Y-) if the spacer unit is present, and the amino acid unit (-W-) connects the extender unit to the cytotoxic agent if the spacer unit is not present.
As previously mentioned, -Ww-may be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit.
In some embodiments, the amino acid unit may comprise an amino acid residue such as, but not limited to, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, arginine protected with a methanesulfonyl group or a nitro group, histidine, ornithine protected with an acetyl or formyl group, and citrulline protected with an acetyl or formyl group. Exemplary amino acid linker components preferably include di-, tri-, tetra-, or penta-peptides.
Exemplary dipeptides include: Val-Cit, Ala-Val, Lys-Lys, Cit-Cit, Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg.
Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.
Exemplary tetrapeptides include: Gly-Phe-Leu-Gly (SEQ ID NO.33), Ala-Leu-Ala-Leu (SEQ ID NO. 34).
Exemplary pentapeptides include: Pro-Val-Gly-Val-Val (SEQ ID NO. 35).
Amino acid residues comprising the amino acid linker component include those naturally occurring, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The enzymatic cleavage selectivity of the amino acid linker component for a particular enzyme, such as tumor associated protease, cathepsin B, C and D, or plasmin protease, can be designed and optimized.
The amino acid units of the linker may be enzymatically cleaved by enzymes (including but not limited to tumor-associated proteases) to release the cytotoxic agent.
The enzymatic cleavage selectivity of the amino acid units for a particular tumor-associated protease can be designed and optimized. Suitable units are those whose cleavage is catalyzed by proteases, cathepsins B, C and D, or plasmin.
When present, the spacer unit (-Y-) links the amino acid unit to the cytotoxic agent. Spacer elements are generally of two types: self-removing (self-removing) and non-self-removing (non self-removing). A non self-immolative spacer is one in which some or all of the spacer remains bound to the cytotoxic agent after the amino acid unit is cleaved enzymatically from the immunoconjugate. Examples of non self-removing spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units. To release the cytotoxic agent, a separate hydrolysis reaction should be undertaken within the target cell to cleave the glycine-drug unit bond.
In another embodiment, the non self-eliminating spacer unit (-Y-) is-Gly-.
In one embodiment, the immunoconjugate lacks a spacer unit (y-0). Alternatively, immunoconjugates comprising self-immolative spacer units can release cytotoxic agents without the need for a separate hydrolysis step. In these embodiments, -Y-is a p-aminobenzyl alcohol (PAB) unit linked to-Ww-through the nitrogen atom of the PAB group, and is directly linked to-D through a carbonate, carbamate, or ether group.
Other examples of self-eliminating spacers include, but are not limited to, aromatic compounds that are electronically equivalent to PAB groups, such as 2-aminoimidazole-5-methanol derivatives and ortho or para-aminobenzyl acetals. Upon hydrolysis of the amide bond, spacers can be used which readily undergo cyclization, such as substituted and unsubstituted 4-aminobutanoic acid amides, appropriately substituted bicyclo [2.2.1] and bicyclo [2.2.2] ring systems, and 2-aminophenylpropionic acid amides.
In an alternative embodiment, the spacer unit is a branched bis (hydroxymethyl) styrene (BHMS) unit, which can be used to incorporate additional cytotoxic agents.
Finally, the invention relates to an immunoconjugate for use in the treatment of cancer as described above.
The cancer may preferably be selected from Axl-associated cancers comprising tumour cells expressing or overexpressing all or part of the protein Axl at their surface.
More specifically, the cancer is breast cancer, colon cancer, esophageal cancer, hepatocellular cancer, gastric cancer, glioma, lung cancer, melanoma, osteosarcoma, ovarian cancer, prostate cancer, rhabdomyosarcoma, renal cancer, thyroid cancer, endometrial cancer, and any drug resistance phenomenon. Another object of the invention is a pharmaceutical composition comprising the immunoconjugate described in the specification.
More specifically, the invention relates to a pharmaceutical composition comprising an immunoconjugate of the invention together with at least one excipient and/or a pharmaceutically acceptable carrier.
In the present description, the expression "pharmaceutically acceptable carrier" or "excipient" is intended to mean a compound or a combination of compounds which enters into the pharmaceutical composition without causing secondary reactions and which allows, for example, to facilitate the administration of the active compound, to increase its lifetime and/or efficacy in vivo, to increase its solubility in solution or to improve its preservation. Such pharmaceutically acceptable carriers and excipients are well known and will be adjusted by those skilled in the art as a function of the nature of the active compound selected and the mode of administration.
Preferably, these immunoconjugates are administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subcutaneous route, or by the oral route. In a more preferred manner, the composition comprising the immunoconjugate according to the invention will be administered several times in a sequential manner.
Its mode of administration, dosage plan and optimal pharmaceutical form may be determined according to criteria that are usually taken into account when establishing a treatment suitable for the patient, such as, for example, the age or weight of the patient, the severity of his/her general condition, the tolerance to the treatment and the side effects noted.
Other features and advantages of the present invention are further presented in the following description with examples and figures, the figures of which are shown below.