Humanized antibodies against CD269 (BCMA)
11820829 · 2023-11-21
Assignee
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K2317/41
CHEMISTRY; METALLURGY
C07K2299/00
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C12N5/10
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
A61K47/6849
HUMAN NECESSITIES
C12N15/00
CHEMISTRY; METALLURGY
A61K47/6867
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/2878
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K47/68
HUMAN NECESSITIES
C12N15/00
CHEMISTRY; METALLURGY
Abstract
A nucleic acid molecule encoding an antibody or antibody fragment, wherein the antibody or antibody fragment binds an epitope of the extracellular domain of CD269 (BCMA), a host cell comprising the nucleic acid molecule and a composition comprising the host cell.
Claims
1. A nucleic acid molecule encoding an antibody or antibody fragment, wherein the antibody or antibody fragment comprises: (a) a VH domain that comprises: CDR1 sequence RYWIS (SEQ ID NO: 18) or RYWFS (SEQ ID NO: 19); CDR2 sequence EINPNSSTINYAPSLKDK (SEQ ID NO: 20) or EINPSSSTINYAPSLKDK (SEQ ID NO: 21); and CDR3 sequence SLYYDYGDAYDYW (SEQ ID NO: 22); and (b) a VL domain that comprises: CDR1 sequence KASQSVX.sub.1X.sub.2NVA (SEQ ID NO: 23), wherein X.sub.1X.sub.2 is ES; CDR2 sequence SASLRFS (SEQ ID NO: 24); and CDR3 sequence QQYNNYPLTFG (SEQ ID NO: 25), wherein said antibody or antibody fragment binds an epitope of the extracellular domain of CD269 (BCMA).
2. The nucleic acid molecule of claim 1, wherein the antibody or antibody fragment comprises a VH domain that comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
3. The nucleic acid molecule of claim 1, wherein the VL domain comprises the sequence of SEQ ID NO: 14.
4. The nucleic acid molecule of claim 1, wherein the antibody binds an epitope of the N-terminus of CD269, wherein the epitope consists of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 or 32 of SEQ ID NO: 39.
5. The nucleic acid molecule of claim 1, wherein the antibody binding to CD269 (BCMA) disrupts BAFF-CD269 and/or APRIL-CD269 interaction.
6. The nucleic acid molecule of claim 1, wherein the VH domain has at least 80% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDR sequences are those recited in claim 1.
7. The nucleic acid molecule of claim 1, wherein the VH domain has at least 90% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDR sequences are those recited in claim 1.
8. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a sequence selected from SEQ ID NO: 33, 34 or 36.
9. A host cell comprising a nucleic acid molecule encoding an antibody or antibody fragment, wherein the antibody or antibody fragment comprises: (a) a VH domain that comprises: CDR1 sequence RYWIS (SEQ ID NO: 18) or RYWFS (SEQ ID NO: 19); CDR2 sequence EINPNSSTINYAPSLKDK (SEQ ID NO: 20) or EINPSSSTINYAPSLKDK (SEQ ID NO: 21); and CDR3 sequence SLYYDYGDAYDYW (SEQ ID NO: 22); and (b) a VL domain that comprises: CDR1 sequence KASQSVX.sub.1X.sub.2NVA (SEQ ID NO: 23), wherein X.sub.1X.sub.2 is ES; CDR2 sequence SASLRFS (SEQ ID NO: 24); and CDR3 sequence QQYNNYPLTFG (SEQ ID NO: 25), wherein said antibody or antibody fragment binds an epitope of the extracellular domain of CD269 (BCMA).
10. The host cell of claim 9, wherein the antibody or antibody fragment comprises a VH domain that comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
11. The host cell of claim 9, wherein the VL domain comprises the sequence of SEQ ID NO: 14.
12. The host cell of claim 9, wherein the antibody binds an epitope of the N-terminus of CD269, wherein the epitope consists of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27 or 32 of SEQ ID NO: 39.
13. The host cell of claim 9, wherein the antibody binding to CD269 (BCMA) disrupts BAFF-CD269 and/or APRIL-CD269 interaction.
14. The host cell of claim 9, wherein the VH domain has at least 80% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDR sequences are those recited in claim 9.
15. The host cell of claim 9, wherein the VH domain has at least 90% sequence identity to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and wherein the CDR sequences are those recited in claim 9.
16. The host cell of claim 9, wherein the nucleic acid molecule comprises a sequence selected from SEQ ID NO: 33, 34 or 36.
17. The host cell of claim 9, wherein the cell expresses the antibody and the antibody is glycosylated.
18. The host cell of claim 17, wherein the antibody comprises the sequence of SEQ ID NO: 29, and wherein the antibody comprises a glycan that is an N-linked oligosaccharide chain at Asn297 of the heavy chain consisting of the sequence of SEQ ID NO: 29.
19. A composition comprising the host cell of claim 9 and a pharmaceutically acceptable carrier.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The invention is demonstrated by way of the example by the examples and figures disclosed herein. The figures provided herein represent particular embodiments of the invention and are not intended to limit the scope of the invention. The figures are to be considered as providing a further description of possible and potentially preferred embodiments that enhance the technical support of one or more non-limiting embodiments.
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DETAILED DESCRIPTION OF THE INVENTION
(15) As used herein, an “antibody” generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Where the term “antibody” is used, the term “antibody fragment” may also be considered to be referred to. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (L) (about 25 kD) and one “heavy” (H) chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms “variable light chain” and “variable heavy chain” refer to these variable regions of the light and heavy chains respectively. Optionally, the antibody or the immunological portion of the antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
(16) The antibodies of the invention are intended to bind against mammalian, in particular human, protein targets. The use of protein names may correspond to either mouse or human versions of a protein.
(17) “Specific binding” is to be understood as via one skilled in the art, whereby the skilled person is clearly aware of various experimental procedures that can be used to test binding and binding specificity. Some cross-reaction or background binding may be inevitable in many protein-protein interactions; this is not to detract from the “specificity” of the binding between antibody and epitope. The term “directed against” is also applicable when considering the term “specificity” in understanding the interaction between antibody and epitope.
(18) Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single variable fragments (ssFv), single domain antibodies (such as VHH fragments from nanobodies), single chain fragments (scFv), Fab fragments, F(ab′).sub.2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies and epitope-binding fragments or combinations thereof of any of the above, provided that they retain the original binding properties. Also mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention. The immunoglobulin molecules of the invention can be of any class (i.e. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Thus, the term antibody, as used herein, also includes antibodies and antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
(19) Humanized antibody comprising one or more CDRs of antibodies of the invention or one or more CDRs derived from said antibodies can be made using any methods known in the art. For example, four general steps may be used to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process (3) the actual humanizing methodologies/techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.
(20) The term humanized antibody means that at least a portion of the framework regions, and optionally a portion of CDR regions or other regions involved in binding, of an immunoglobulin is derived from or adjusted to human immunoglobulin sequences. The humanized, chimeric or partially humanized versions of the mouse monoclonal antibodies can, for example, be made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (Queen et al., 1989; WO 90/07861). Alternatively the monoclonal antibodies used in the method of the invention may be human monoclonal antibodies. Human antibodies can be obtained, for example, using phage-display methods (WO 91/17271; WO 92/01047).
(21) As used herein, humanized antibodies refer also to forms of non-human (e.g. murine, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
(22) As used herein, human or humanized antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art or disclosed herein. Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly share the useful functional properties of the mouse antibodies to a large extent. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillar and/or non-fibrillar polypeptides or fragments thereof as an affinity reagent. Monoclonal antibodies can be obtained from serum according to the technique described in WO 99/60846.
(23) The present invention further relates to the use of the antibodies, or fragments thereof, as described herein, for example the variable regions, in recognition molecules or affinity reagents that are suitable for selective binding to a target. The affinity reagent, antibody or fragment thereof according to the invention may be PEGylated, whereby PEGylation refers to covalent attachment of polyethylene glycol (PEG) polymer chains to the inventive antibody. PEGylation may be routinely achieved by incubation of a reactive derivative of PEG with the target molecule. PEGylation to the antibody can potentially mask the agent from the hosts immune system, leading to reduced immunogenicity and antigenicity or increase the hydrodynamic size of the agent which may prolong its circulatory time by reducing renal clearance.
(24) A variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.
(25) In some embodiments, the invention provides an antibody, which comprises at least one CDR, at least two, at least three, or more CDRs that are substantially identical to at least one CDR, at least two, at least three, or more CDRs of the antibody of the invention. Other embodiments include antibodies which have at least two, three, four, five, or six CDR(s) that are substantially identical to at least two, three, four, five or six CDRs of the antibodies of the invention or derived from the antibodies of the invention. In some embodiments, the at least one, two, three, four, five, or six CDR(s) are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two or three CDRs of the antibody of the invention. It is understood that, for purposes of this invention, binding specificity and/or overall activity is generally retained, although the extent of activity may vary compared to said antibody (may be greater or lesser).
(26) The half life and cytotoxic potential of an antibody are dependent primarily on the interaction of the Fc-domain with different Fc-gamma-receptors. In the case of the antibody half life, the neonatal Fc receptor (FcRn) plays a major role. This receptor is expressed on several cell types and tissues such as monocytes and vascular endothelia cells that are able to take up serum proteins into their recycling endosomes. In the endosomes, the pH is decreased to approximately 6 and under these conditions the antibodies are able to bind to FcRn. This interaction protects the antibodies from degradation until they are again released into the blood where the physiological pH disrupts the binding to the receptor (Roopenian and Akilesh (2007) Nat Rev Immunol 7:715-725). The higher the affinity of the antibody to the FcRn at pH 6, the greater the half life of that antibody. Fc-fragment mutations known to stabilize this interaction are summarised in Presta (2008, Curr Opin Immunol 20:460-470).
(27) Therapeutic antibodies can act through several mechanisms upon binding to their target. The binding itself can trigger signal transduction, which can lead to programmed cell death (Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25). It can also block the interaction of a receptor with its ligand by either binding to the receptor or the ligand. This interruption can cause apoptosis if signals important for survival are affected (Chiu et al. (2007) Blood 109:729-739). With regard to cell-depletion there are two major effector mechanisms known. The first is the complement-dependent cytotoxicity (CDC) towards the target cell. There are three different pathways known. However, in the case of antibodies the important pathway for CDC is the classical pathway which is initiated through the binding of Clq to the constant region of IgG or IgM (Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768).
(28) The second mechanism is called antibody-dependent cellular cytotoxicity (ADCC). This effector function is characterized by the recruitment of immune cells which express Fc-receptors for the respective isotype of the antibody. ADCC is largely mediated by activating Fc-gamma receptors (FcγR) which are able to bind to IgG molecules either alone or as immune complexes. Mice exhibit three (FcγRI, FcγRIII and FcγRIV) and humans five (FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA and FcγRIIIB) activating Fcγ-receptors. These receptors are expressed on innate immune cells like granulocytes, monocytes, macrophages, dendritic cells and natural killer cells and therefore link the innate with the adaptive immune system. Depending on the cell type there are several modes of action of FcgR-bearing cells upon recognition of an antibody-marked target cell. Granulocytes generally release vasoactive and cytotoxic substances or chemoattractants but are also capable of phagocytosis. Monocytes and macrophages respond with phagocytosis, oxidative burst, cytotoxicity or the release of pro-inflammatory cytokines whereas Natural killer cells release granzymes and perforin and can also trigger cell death through the interaction with FAS on the target cell and their Fas ligand (Nimmerjahn and Ravetch (2008) Nat Rev Immunol 8:34-47; Wang and Weiner (2008) Expert Opin Biol Ther 8:759-768; Chavez-Galan et al. (2009) Cell Mol Immunol 6:15-25).
(29) The antibody-dependent cellular cytotoxicity (ADCC) can also be improved by strengthening the binding of the Fc-domain to activating Fc-gamma receptors (FcγR). This can also be achieved through mutations in the Fc-gamma domain as summarized in Presta (2008, Curr Opin Immunol 20:460-470).
(30) Another way to change the ADCC is manipulation of the sugar moiety present on each IgG at Asn297. Defucolylation and removal of sialic acid from the end of the sugar molecules are known to increase the cytotoxic potential of an antibody (Anthony and Ravetch (2010) J Clin Immunol 30 Suppl 1:S9-14).
(31) Sequence variants of the claimed nucleic acids, proteins and antibodies, for example defined by the claimed % sequence identity, that maintain the said properties of the invention are also included in the scope of the invention. Such variants, which show alternative sequences, but maintain essentially the same binding properties, such as target specificity, as the specific sequences provided are known as functional analogues, or as functionally analogous. Sequence identity relates to the percentage of identical nucleotides or amino acids when carrying out a sequence alignment.
(32) It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology or sequence identity to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in sequence that fall under the described sequence identity are also encompassed in the invention.
(33) Protein sequence modifications, which may occur through substitutions, are also included within the scope of the invention. Substitutions as defined herein are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein which contains a different amino acid sequence than the primary protein, preferably without significantly altering the function of the protein. Like additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function. This is particularly true when the modification relates to a “conservative” amino acid substitution, which is the substitution of one amino acid for another of similar properties. Such “conserved” amino acids can be natural or synthetic amino acids which because of size, charge, polarity and conformation can be substituted without significantly affecting the structure and function of the protein. Frequently, many amino acids may be substituted by conservative amino acids without deleteriously affecting the protein's function.
(34) In general, the non-polar amino acids Gly, Ala, Val, Ile and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu, represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups.
(35) Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in the table immediately below, or as further described below in reference to amino acid classes, may be introduced and the products screened.
(36) Potential Amino Acid Substitutions:
(37) TABLE-US-00013 Preferred Original conservative Examples of residue substitutions exemplary substitutions Ala (A) Val Val; Leu; Ile Asg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn, Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala; Norleucine
(38) Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
(39) Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexyl alanine which are neutral non-polar analogs; citrulline and methionine sulfoxide which are neutral non-polar analogs, phenylglycine which is an aromatic neutral analog; cysteic acid which is a negatively charged analog and ornithine which is a positively charged amino acid analog. Like the naturally occurring amino acids, this list is not exhaustive, but merely exemplary of the substitutions that are well known in the art.
(40) The antibodies of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antibody of the invention. An expression vector or recombinant plasmid is produced by placing these coding sequences for the antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antibody light or heavy chain. In certain embodiments this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the antibody may reside on a single vector.
(41) A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention. The antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antibodies.
(42) Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially available. The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
(43) The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antibodies of the present invention. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional.
(44) Suitable host cells or cell lines for the expression of the antibodies of the invention include mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns. Alternatively, other prokaryotic or eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art.
(45) In accordance with the present invention there is provided a method of producing an anti-CD269-antibody of the present invention which binds to and neutralises the activity of human CD269 which method comprises the steps of; providing a first vector encoding a heavy chain of the antibody; providing a second vector encoding a light chain of the antibody; transforming a mammalian host cell (e.g. CHO) with said first and second vectors; culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said host cell into said culture media; recovering the secreted antibody of step (d). Once expressed, the antibody can be assessed for the desired binding properties using methods as described herein.
(46) The invention encompasses immunoconjugates (interchangeably referred to as “antibody-drug conjugates” or “ADCs”) comprising an antibody according to the invention as herein described including, but not limited to, an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, such as for the Anti-CD269 Antibody-Drug Conjugates of the present invention, are well-known. (See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1995); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89/12624.)
(47) Typically, the ADC or ADC derivative comprises a linker region between the therapeutic agent and the anti-CD269 antibody or derivative thereof. As noted supra, in typical embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment. For example, in some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art (See for example Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
(48) Typically, the linker is not substantially sensitive to the extracellular environment. In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (i.e., in the milieu of the linker-therapeutic agent moiety of the ADC or ADC derivate as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the therapeutic agent and the anti-CD269 antibody or derivative thereof (i.e., in the milieu of the ADC or ADC derivative as described herein). A variety of linkers that can be used with the present compositions and methods are described in WO 2004010957 entitled “Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease” filed Jul. 31, 2003, and U.S. Provisional Application No. 60/400,403, entitled “Drug Conjugates and their use for treating cancer, an autoimmune disease or an infectious disease”, filed Jul. 31, 2002 (the disclosure of which is incorporated by reference herein).
(49) In certain embodiments, an immunoconjugate comprises an antibody as described herein, including but not limited to, an antibody and a chemotherapeutic agent or other toxin. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies.
(50) Antibodies or fragments thereof of the present invention may also be conjugated to one or more toxins, including, but not limited to, a calicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity. Suitable cytotoxic agents include, but are not limited to, an auristatin including dovaline-valine-dolaisoleunine-dolaproine-phenylalanine (MMAF) and monomethyl auristatin E (MMAE) as well as ester forms of MMAE, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, including paclitaxel and docetaxel, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, DM-4, netropsin. Other suitable cytotoxic agents include anti-tubulin agents, such as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolastatin. Antitubulin agent include dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-iamine (AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, DM-4 or eleutherobin.
(51) In some embodiments, the immunoconjugate comprises an antibody conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin (which are pentapeptide derivatives of dolastatins) drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172). Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, disclosed in “Monomethylvaline Compounds Capable of Conjugation to Ligands,” U.S. Pat. No. 7,498,298. As used herein, the abbreviation “MMAE” refers to monomethyl auristatin E. As used herein the abbreviation “MMAF” refers to dovaline-valine-dolaisoleuine-dolaproine-phenylalanine.
(52) Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see E. Schroder and K. Lubke, “The Peptides,” volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
(53) Maytansinoids may be used as an active agent coupled to the antibody or fragment thereof according to the invention. Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Highly cytotoxic maytansinoid drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources.
(54) Selected examples of the calicheamicin family of antibiotics may be used as an active agent coupled to the antibody or fragment thereof according to the invention. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296. Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
(55) Other antitumor agents that can be conjugated to the antibodies include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296). The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase). For selective destruction of the tumor, the antibody may comprise a highly radioactive atom.
(56) A pharmaceutically acceptable carrier in the sense of the present invention may be any non-toxic material that does not significantly interfere in a detrimental sense with the effectiveness of the biological activity of the antibodies of the present invention. Evidently, the characteristics of the carrier will depend on the route of administration. Such a composition may contain, in addition to the active substance and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intra-muscular administration and formulation.
(57) The medicament, otherwise known as a pharmaceutical composition, containing the active ingredient (antibody or antibody fragment) may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated. The present invention also refers to a pharmaceutical composition for topical application, oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. A skilled person is aware of the carriers and additives required for particular application forms.
(58) When a therapeutically effective amount of the active substance (antibody or antibody fragment) of the invention is administered by intravenous, cutaneous or subcutaneous injection, the active substance may be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
(59) The invention also relates to administration of a therapeutically relevant amount of antibody as described herein in the treatment of a subject who has the medical disorders as disclosed herein. As used herein, the term “therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit. The amount of active substance in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Larger doses may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
(60) The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the active substance, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
(61) The dose of the antibody administered evidently depends on numerous factors well-known in the art such as, e.g., the chemical nature and pharmaceutical formulation of the antibody, and of body weight, body surface, age and sex of the patient, as well as the time and route of administration. For an adult, the dose may exemplarily be between 0.001 μg and 1 g per day, preferably between 0.1 μg and 100 mg per day, more preferably between 1 μg and 100 mg per day, even more preferably between 5 μg and 10 mg per day. In a continuous infusion, the dose may exemplarily be between 0.01 μg and 100 mg, preferably between 1 μg and 10 mg per kilogram body mass per minute.
(62) In another aspect of the present invention there is provided an antibody according to the invention as herein described for use in the treatment of a B-cell mediated or plasma cell mediated disease or antibody mediated disease or disorder selected from Multiple Myeloma (MM), chronic lymphocytic leukemia (CLL), Non-secretory multiple myeloma, Smoldering multiple myeloma, Monoclonal gammopathy of undetermined significance (MGUS), Solitary plasmacytoma (Bone, Extramedullar), Lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, Plasma cell leukemia, Primary Amyloidosis (AL), Heavy chain disease, Systemic lupus erythematosus (SLE), POEMS syndrome/osteosclerotic myeloma, Type I and II cryoglobulinemia, Light chain deposition disease, Goodpasture's syndrome, Idiopathic thrombocytopenic purpura (ITP), Acute glomerulonephritis, Pemphigus and Pemphigoid disorders, and Epidermolysis bullosa acquisita; or any Non-Hodgkin's Lymphoma B-cell leukemia or Hodgkin's lymphoma (HL) with BCMA expression or any diseases in which patients develop neutralising antibodies to recombinant protein replacement therapy wherein said method comprises the step of administering to said patient a therapeutically effective amount of the antibody as described herein.
(63) B-cell disorders can be divided into defects of B-cell development/immunoglobulin production (immunodeficiencies) and excessive/uncontrolled proliferation (lymphomas, leukemias). As used herein, B-cell disorder refers to both types of diseases, and methods are provided for treating B-cell disorders with an antibody.
(64) In one aspect of the present invention the disease is Multiple Myeloma.
(65) Use of the antibody as described herein in the manufacture of a medicament for the treatment of diseases and disorders as described herein is also provided.
(66) For example in one aspect of the invention there is provided the use of the antibody as described herein for use in the treatment or prophylaxis of diseases and disorders responsive to modulation (such as inhibiting or blocking) of the interaction between BCMA and the ligands BAFF and APRIL.
(67) In one embodiment of the invention the isolated antibody or antibody fragment is intended for use in the treatment of B lymphocyte cancers, such as Hodgkin's lymphoma.
(68) In one embodiment of the invention the isolated antibody or antibody fragment is intended for use in the treatment of an autoimmune disease, such as a medical disorder associated with inflammation, preferably autoimmune disease with an inflammatory component, whereby the autoimmune disease is selected from Takayasu Arteritis, Giant-cell arteritis, familial Mediterranean fever, Kawasaki disease, Polyarteritis nodosa, cutanous Polyarteritis nodosa, Hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitidies, Churg-Strauss syndrome, microscopic polyangiitis, Vasculitis of connective tissue diseases, Hennoch-Schõnlein purpura, Cryoglobulinemic vasculitis, Cutaneous leukocytoclastic angiitis, Tropical aortitis, Sarcoidosis, Cogan's syndrome, Wiskott-Aldrich Syndrome, Lepromatous arteritis, Primary angiitis of the CNS, Thromboangiitis obliterans, Paraneoplastic ateritis, Urticaria, Dego's disease, Myelodysplastic syndrome, Eythema elevatum diutinum, Hyperimmunoglobulin D, Allergic Rhinitis, Asthma bronchiale, chronic obstructive pulmonary disease, periodontitis, Rheumatoid Arthritis, atherosclerosis, Amyloidosis, Morbus Chron, Colitis ulcerosa, Autoimmune Myositis, Diabetes mellitus, Multiple sclerosis, Guillain-Barre Syndrome, histiocytosis, Osteoarthritis, atopic dermatitis, periodontitis, chronic rhinosinusitis, Psoriasis, psoriatic arthritis, Microscopic colitis, Pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome, Lupus erythematosus, aplastic anemia, Osteomyelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, Idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, Autoimmunthrombocytopenia, Autoimmunneutropenia, Autoimmunhemolytic anemia, Autoimmunlymphocytopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's Thyroiditis, atropic thyroiditis, Graves disase, Autoimmune polyglandular syndrome, Autoimmune Addison Syndrome, Pemphigus vulgaris, Pemphigus foliaceus, Dermatitis herpetiformis, Autoimmune alopecia, Vitiligo, Antiphospholipid syndrome, Myasthenia gravis, Stiff-man syndrome, Goodpasture's syndrome, Sympathetic ophthalmia, Folliculitis, Sharp syndrome and/or Evans syndrome, in particular hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, preferably rheumatoid arthritis or multiple sclerosis.
SEQUENCES
(69) Preferred Antibody Sequences of the Invention:
(70) TABLE-US-00014 SEQ ID No. Sequence Description SEQ ID No. 1 QVQLQQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVR HC (VH) mouse RAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAK NTLYLQMSKVRSEDTALYYCASLYYDYGDAMDYWGQGT SVTVSS SEQ ID No. 2 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYWMSWVR HC partially QAPGKGLEWVGEINPDSSTINYAPSLKGRFTISRDNAK humanized NTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGT LVTVSS SEQ ID No. 3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR hHC01 QAPGKGLVWVGEINPDSSTINYAPSLKDKFTISRDNAK NTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGT LVTVSS SEQ ID No. 4 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWX.sub.1SWV hHC02 RQAPGKGLVWVGEINPX.sub.2X.sub.3STINYAPSLKDKFTISRD NAKNTLYLQMNSLRAEDTAVYYCASLYX.sub.4DYGDAX.sub.5DY WGQGTLVTVSS Wherein X.sub.1: I, F, L, V, Y. C, G, A, S, T, preferably I or F; X.sub.2X.sub.3: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS; X.sub.4: Y, L, A, V, F, I, W, preferably Y; and/or X.sub.5: Y, L, F, I, V, A, C, preferably Y SEQ ID No. 5 EVQLVESGGGLVQPGGSLRLSGAASGFTFSRYX.sub.1MX.sub.2W hHC03 VRQAPGKGLVX.sub.3VGX.sub.4INPDSSTINYAPSLKDKFTISR DNAKNTLYLQMNSLRAEDTAVYYCASX.sub.5X.sub.6X.sub.7DYGDX.sub.8M DYWGQGTLVTVSS Wherein X.sub.1: W, F, Y, preferred W; X.sub.2: S, T, N, Q, D, E, preferred S; X.sub.3: W, F, Y, preferred W; X.sub.4: E, Q, preferred E; X.sub.5: L, I, V, G, A, preferred L; X.sub.6: Y, X, preferred Y; X.sub.7: Y, F, L, I, V, M, preferred Y; and/or X.sub.8: A, G, V, preferred A SEQ ID No. 6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC04 ISWVRQAPGKGLVWVGEINPNSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC05 FSWVRQAPGKGLVWVGEINPNSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC06 ISWVRQAPGKGLVWVGEINPSSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYW hHC07 FSWVRQAPGKGLVWVGEINPSSSTINYAPSLKD KFTISRDNAKNTLYLQMNSLRAEDTAVYYCASL YYDYGDAYDYWGQGTLVTVSS SEQ ID No. 43 DIVMTQSQRFMTTSVGDRVSVTCKASQSVDSNV LC (VL) mouse AWYQQKPRQSPKALIFSASLRFSGVPARFTGSG SGTDFTLTISNLQSEDLAEYFCQQYNNYPLTFG AGTKLELKR SEQ ID No. 10 DIVMTQSPATLSVSVGDEVTLTCKASQSVDSNVAWYQQ LC partially KPGQAPKLLIYSDDLRFSGVPARFSGSGSGTDFTLTIS humanized SLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR SEQ ID No. 11 EIVMTQSPATLSVSPGERATLSCKASQSVDSNVAWYQQ hLC01 KPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS SLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR SEQ ID No. 12 EIVMTQSPATLSVSPGERATLSCKASQSVX.sub.1X.sub.2NVAWY hLC02 QQKPGQAPRALIYSASLRFSGIPARESGSGSGTEFTLT ISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELKR Wherein: X.sub.1X.sub.2: ES, SS, TS, QS, HS, DH, preferably ES. SEQ ID No. 13 EIVMTQSPATLSVSPGERATLSCKASQSVDX.sub.1X.sub.2VX.sub.3W hLC03 X.sub.4QQKPGQAPRALIX.sub.5X.sub.6AX.sub.7X.sub.8RX.sub.9SGIPARFSG5X.sub.10X.sub.11 GTEFTLTISSLQSEDFAVYYCX.sub.12QX.sub.13NNX.sub.14PX.sub.15TFG AGTKLELKR Wherein: X.sub.1: S, H, T, N, D, Q; X.sub.2: N, E, Q; X.sub.3: A, G, V, S, T, L, I; X.sub.4: Y, F, L, I, V, A, G; X.sub.5: Y, F, L; X.sub.6: S, T; X.sub.7: S, T, D, N, H, E, Q; X.sub.8: L, V, I, M; X.sub.9: F, L, I, V, Y, M; X.sub.10: G, X; X.sub.11: S, X; X.sub.12: Q, V, L, I, M; X.sub.13: Y, F, L, I, Q; X.sub.14: Y, F, R, Q, K; and/or X.sub.15: L, I, V, F SEQ ID No. 14 EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQ hLC04 KPGQAPRALIYSASLRFSGIPARESGSGSGTEFTLTIS SLQSEDFAVYYCQQYNNYPLIFGAGTKLELKR SEQ ID No. 15 RYWX.sub.1S H-CDR1 PTM Wherein: X.sub.1: I, F, L, V, Y. C, G, A, S, T, preferably I or F SEQ ID No. 16 EINPX.sub.2X.sub.3STINYAPSLKDK H-CDR2 PTM Wherein: X.sub.2X.sub.3: SS, NS, TS, GS, KS, RS, SD, SN, DE, preferably SS SEQ ID No. 17 SLYX.sub.4DYGDAX.sub.5DYW H-CDR3 PTM Wherein: X.sub.4: Y, L, A, V, F, I, W, preferably Y; and/or X.sub.5: Y, L, F, I, V, A, C, preferably Y SEQ ID No. 18 RYWIS H-CDR1 PTM a SEQ ID No. 19 RYWFS H-CDR1 PTM b SEQ ID No. 20 EINPNSSTINYAPSLKDK H-CDR2 PTM a SEQ ID No. 21 EINPSSSTINYAPSLKDK H-CDR2 PTM b SEQ ID No. 22 SLYYDYGDAYDYW H-CDR3 PTM a SEQ ID No. 23 KASQSVX.sub.1X.sub.2NVA L-CDR1 PTM Wherein: X.sub.1X.sub.2: ES, SS, TS, QS, HS, DH, preferably ES SEQ ID No. 24 SASLRFS L-CDR2 PTM SEQ ID No. 25 QQYNNYPLTFG L-CDR3 PTM SEQ ID No. 26 KASQSVDSNVA L-CDR1 PTM a SEQ ID No. 27 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR Full length QAPGKGLVWVGEINPDSSTINYAPSLKDKFTISRDNAK humanized HC NTLYLQMNSLRAEDTAVYYCASLYYDYGDAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 28 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVR Full length QAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAK humanized HC with NTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGT PTM mutations 1 LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY (FSY) FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 29 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWISWVR Full length QAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAK humanized HC with NTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGT PTM mutations 2 LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY (ISY) FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGOPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 30 EIVMTQSPATLSVSPGERATLSCKASQSVDSNVAWYQQ Full length KPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS humanized LC SLQSEDFAVYYCQQYNNYPLTFGAGTKLELKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 31 EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQ Full length KPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTIS humanized LC with SLQSEDFAVYYCQQYNNYPLTFGAGTKLELKRTVAAPS PTM mutations VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC
(71) Preferred Nucleotide Sequences
(72) TABLE-US-00015 SEQ ID No. 32 GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full length GTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HC TCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCT TAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGG TATTGGATGAGCTGGGTGAGACAGGCTCCAGGGAAAGGTC TGGTGTGGGTAGGGGAGATAAACCCCGATAGCAGCACGAT CAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCC CGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATT CCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAG TCTGTACtacgactATGGGGACGCAATGGACTATTGGGGA CAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 33 GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full length GTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HC with TCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCT PTM mutations 1 TAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGG TATTGGTTCAGCTGGGTGAGACAGGCTCCAGGGAAAGGTC TGGTGTGGGTAGGGGAGATAAACCCCAGCAGCAGCACGAT CAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCC CGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATT CCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAG TCTGTACTACGACTATGGGGACGCATACGACTATTGGGGA CAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 34 GAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTA Full length GTAGCAACTGCAACCGGTGTCCACAGTGAAGTGCAGCTGG humanized HC with TCGAATCTGGAGGAGGCCTGGTTCAGCCTGGTGGCAGCCT PTM mutations 2 TAGGCTCTCTTGTGCAGCCTCTGGCTTTACCTTCTCACGG TATTGGaTCAGCTGGGTGAGACAGGCTCCAGGGAAAGGTC TGGTGTGGGTAGGGGAGATAAACCCCAGCAGCAGCACGAT CAACTATGCTCCGTCACTGAAAGACAAGTTCACCATTTCC CGCGATAATGCCAAGAACACTCTCTACTTGCAGATGAATT CCCTTCGAGCCGAGGATACAGCGGTGTACTACTGCGCCAG TCTGTACTACGACTATGGGGACGCATACGACTATTGGGGA CAAGGCACACTGGTGACTGTTAGCTCCGCGTCGACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCCCCGGGTAAATGAGTGCGACGGCCGGGCGGCGG CGGCGGATCC SEQ ID No. 35 GAATTCCACCATGGGATGGtcATGTATCATCCTTTTTCTA Full length GTAGCAACTGCAACCGGTGTACACTCCGAGATCGTGATGA humanized LC CCCAGTCTCCTGCTACCCTGAGCGTTTCTCCCGGTGAAAG GGCCACACTCAGCTGCAAAGCCTCTCAAAGCGTGGACAGC AATGTCGCCTGGTATCAGCAGAAACCTGGCCAAGCTCCGA GAGCACTGATCTATTCCGCGTCATTGCGCTTTTCCGGCAT ACCAGCACGGTTTAGTGGCTCAGGGAGTGGGACTGAGTTC ACTCTGACGATTAGCTCCCTTCAGTCAGAGGATTTCGCCG TGTACTACTGTCAGCAGTACAACAACTATCCCCTCACATT CGGAGCTGGAACCAAGCTGGAACTGAAGCGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGT TGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGATCC SEQ ID No. 36 GAATTCCACCATGGGATGGtcATGTATCATCCTTTTTCTA Full length GTAGCAACTGCAACCGGTGTACACTCCGAGATCGTGATGA humanized LC with CCCAGTCTCCTGCTACCCTGAGCGTTTCTCCCGGTGAAAG PTM mutations GGCCACACTCAGCTGCAAAGCCTCTCAAAGCGTGGAGAGC AATGTCGCCTGGTATCAGCAGAAACCTGGCCAAGCTCCGA GAGCACTGATCTATTCCGCGTCATTGCGCTTTTCCGGCAT ACCAGCACGGTTTAGTGGCTCAGGGAGTGGGACTGAGTTC ACTCTGACGATTAGCTCCCTTCAGTCAGAGGATTTCGCCG TGTACTACTGTCAGCAGTACAACAACTATCCCCTCACATT CGGAGCTGGAACCAAGCTGGAACTGAAGCGTACGGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGT TGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGATCC
(73) Preferred Sequences of the Invention Pertaining to CD269 (BMCA):
(74) TABLE-US-00016 SEQ ID No. Sequence Description SEQ ID No. 37 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK GST-BCMA-His WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA WPLQGWQATFGGGDHPPKSDLVPRGSMAGQCSQNEYFDSL LHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNALEH HHHHH SEQ ID No. 38 MAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNA BCMA extracellular SVTNSVKGTNALE domain SEQ ID No. 39 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRY BCMA N-terminus CNASVTNSVKGTNALE sequence SEQ ID No. 40 YFDSLLHACIPCQLRCSSNT BCMA antibody epitope - amino acids 13 to 32 of BCMA
(75) Preferred Generalized Amino Acid Sequences Comprising the Humanized Sequence Modifications:
(76) TABLE-US-00017 SEQ ID No. Sequence Description SEQ ID No. 41 X.sub.1VQLX.sub.2X.sub.3SGGGLVQPGGSLX.sub.4LSCAASGX.sub.5X.sub.6FX.sub.7X.sub.8YWZ.sub.1 General sequence for SWVRX.sub.9APGKGLEWX.sub.10GEINPZ.sub.2SSTINYAPSLKX.sub.11X.sub.12F humanized HC X.sub.13ISRDNAKNTLYLQMX.sub.14X.sub.15X.sub.16RX.sub.17EDTAX.sub.18YYCASLY antibodies comprising YDYGDAZ.sub.3DYWGQGTX.sub.19VTVSS the PTM deletion wherein X1: Q, E; X2: Q, V; X3: Q, E; modifications X4: K, R; X5: I,F; X6: D, T; X7: S, D; X8: R, D; X9: R, Q; X10: I, V; X11: D, G; X12: K, R; X13: I, T; X14: S, N; X15: K, S; X16: V, L; X17: S, A; X18: L, V; X19: S, L; and wherein at least one of Z.sub.1: I or F; Z.sub.2: S and/or Z.sub.3: Y. SEQ ID No. 42 X.sub.1IVMTQSX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7SVGBX.sub.8VX.sub.9X.sub.10TCKASQSVESNV General sequence for AWYQQKPX.sub.11QX.sub.12PKX.sub.13LIX.sub.14SX.sub.15X.sub.16LRFSGVPARFX.sub.17G humanized LC SGSGTDFTLTISX.sub.18LQSEDX.sub.19AX.sub.20YX.sub.21CQQYNNYPLTF antibodies comprising GAGTKLELKR the PTM deletion wherein X1: D, E; X2: Q, P; X3: R, A; modifications X4: F, T; X5: M, L; X6: T, S; X7: T, V; X8: R, E; X9: S, T; X10: V,L; X11: R, G; X12: S, A; X13: A, L; X14: F, Y; X15: A, D; X16: S, D; X17: T, S; X18: N, S; X19: L, F; X20: E, V; X21: F, Y
Examples
(77) The invention is demonstrated by way of the examples disclosed herein. The examples provided herein represent only particular embodiments of the invention and are not intended to limit the scope of the invention. The examples are to be considered as providing a further description of possible and potentially preferred embodiments that enhance the technical support of one or more non-limiting embodiments.
(78) Although the examples with respect to crystallization of the antibody-epitope complex and the in vitro and in vivo anti-tumor effect was carried out using the original chimeric antibody J22.9-xi, the inventors assert that these technical effects are maintained in the humanized variants of the present invention, due to the maintenance of binding characteristics in the humanized variants compared to the original chimeric antibody tested. The data provided from the chimeric antibody is therefore provided as reference material and an indication of the industrial applicability and usefulness of the claimed human variants. Preliminary biological data indicates comparable effects between J22.9-xi and the humanized variants.
(79) Binding and Blocking Characteristics of the J22.9-xi and BCMA Interaction
(80) The novel chimeric antibody (J22.9-xi) binds to the extracellular domain of human CD269 (BCMA, TNFRSF17). This was initially ascertained by ELISA and flow cytometry on the human multiple myeloma cell line MM.1S (
(81) BCMA is known to trigger signals important for the survival of multiple myeloma and plasma cells in vivo through interaction with its ligands BAFF and/or APRIL (Mackay F et al. (2003) Annu Rev Immunol 21:231-264). An in vitro blocking assay was therefore performed with the extracellular domain of human BCMA and recombinant BAFF. The binding of J22.9-xi to BCMA clearly blocks the interaction between the receptor and its ligand BAFF. Using the isotype control antibody instead of J22.9-xi, recombinant BAFF binding to BCMA is unaffected (
(82) The Crystal Structure of the J22.9-xi-Fab-BCMA-Complex Reveals an Extensive Binding Interface with BCMA
(83) Fab fragments prepared from J22.9-xi were crystallized in complex with the purified 46 amino acid residue BCMA extracellular domain and the complex structure solved to 1.9 angstroms resolution. High quality electron density is observable for residues 6 to 41 of BCMA and shows an extensive interaction with J22.9-xi, primarily with the light chain of the antibody (
(84) As can be seen in
(85) TABLE-US-00018 TABLE 1 Amino acid interaction list between heavy chain of J22.9-xi and BCMA. These interaction lists were generated using the software PDBsum (Laskowski R A (2009)). Heavy chain CD269 Trp33 > His19 Glu50 > His19 Leu99 > Leu17 > Leu18 Tyr100 Leu18 Tyr101 > Ala20 > Ile22 > Pro23 Ala106 > Leu18
(86) TABLE-US-00019 TABLE 2 Amino acid interaction list between light chain of J22.9-xi and BCMA. These interaction lists were generated using the software PDBsum (Laskowski R A (2009)). Light chain CD269 Ser31 > Arg27 Thr32 Ala34 > Leu17 Tyr36 > Leu17 Phe49 > Leu18 Asp15 Ser50 > Tyr13 Asp15 Arg27 Ser52 > Arg27 Ser67 > Thr32 Leu53 > Tyr13 Leu26 Arg27 Phe55 > Leu18 Gln89 > Leu17 Tyr91 > Asp15 Ser16 Leu17 Tyr94 > His19 Leu96 > Leu17
(87) TABLE-US-00020 TABLE 3 Interaction list of the residues involved in CD269:APRIL und CD269:BAFF binding (residues NOT directly contacted by J22.9 are underlined). These interaction lists were generated using the software PDBsum (Laskowski R A (2009)). APRIL CD269 Asp121 Leu35 Asp123 Pro33 Pro34 Leu35 Asp164 Asn31 Thr166 Arg27 Ser30 Phe167 Tyr13 Leu18 Ile22 Leu26 Arg27 Asn31 Thr168 Leu18 Leu26 Met169 Leu17 Gly170 Leu17 His19 Gln171 Leu17 Arg186 Leu17 Leu18 His19 Cys187 Leu17 Ile188 Asp15 Leu17 Leu18 Asp196 Leu26 Arg197 Leu26 Tyr199 Leu18 Pro221 Leu17 His19 Arg222 Asp15 Leu17 Arg27 Asn224 Thr32 Lys226 Asn31 His232 His19 BAFF CD269 Tyr22 Ser16 Asp62 Asn31 Lys63 Ser30 Asn31 Thr64 Arg27 Ser30 Asn31 Tyr65 Tyr13 Asp15 Leu18 Ile22 Ala66 Leu17 Met67 Leu17 Gly68 Leu17 Arg90 Leu17 His19 Cys91 Leu17 Ile92 Leu17 Leu18 Glu97 Ser29 Ser30 Asn31 Leu99 Ile22 Leu26 Asn101 Leu18 Pro123 Ser16 Leu17 Arg124 Tyr13 Asp15 Leu17 Arg27 Glu125 Arg27 Thr25 Pro34 Leu35 Asn126 Thr32 Asp132 His19
(88) TABLE-US-00021 TABLE 4 Residues of the CD269 target bound by direct contacts of J22.9, APRIL and/or BAFF. Residues of the CD269 target directly contacted only by J22.9 are underlined (20, 23). Residues of the CD269 target NOT directly contacted by J22.9 are in bold type (30, 31, 33, 34, 35 for APRIL; 25, 29, 30, 31, 34, 35 for BAFF). J22.9: 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, 32 APRIL: 13, 15, 17, 18, 19, 22, 26, 27, 30, 31, 32, 33, 34, 35 BAFF: 13, 15, 16, 17, 18, 19, 22, 25, 26, 27, 29, 30, 31, 32, 34, 35
(89) TABLE-US-00022 TABLE 5 J22.9 Water interactions (J22.9-xi:H2O:CD269). The data in table 5 was generated using the software LigPlot (Wallace and Laskowski, European Bioinformatics Institute). (sc = side chain H-bond; mc = main chain H-bond) Light Chain H.sub.2O# CD269 Ser31 (sc) 285 Thr32 (sc, mc) 285, 286 Arg27 (sc) 285, 286 Ser30 (sc) Ser31 (mc) 283, 284 Arg27 (sc) Asn32 (sc) 105 Asp15 (sc) 105, 284 Arg27 (sc) 56 Ser16 (sc) Tyr36 66, 93, 450 Leu17 (mc) Ser50 (sc) 105 Asp15 (sc) Ser52 (sc) 286 Ser30 (sc) 286 Arg27 (sc) 286, 285 Thr32 (sc, mc) Gly66 (mc) 287 Thr32 (sc) 285, 286 Arg27 (sc) 285, 286 Ser30 (sc) Gln89 (sc) 66, 93, 450 Leu17 (mc) Tyr91 (mc) 282 Ser16 (sc) 282, 281 Ser16 (mc) Tyr94 (sc) 281 Ser16 (mc) 281, 282 Ser16 (sc) Heavy Chain H.sub.2O# CD269 Trp33 (mc) 42, 280 Leu17 (mc) 183, 279, 26 Leu18 (mc) Ser35 (sc) 42, 66, Leu17 (mc) 93, 280, 450 Trp47 (sc) 93, 450 Leu17 (mc) Glu50 (sc) 281 Ser16 (mc) 281, 282 Ser16 (sc) 450 Leu17 (mc) 450, 280 Leu17 (mc) Leu99 (mc) 280 Leu17 (mc) Tyr101 (mc) 26 Leu18 (mc)
(90) Strong Cytotoxic Efficacy of J22.9-xi is Strongly Decreased after Deglycosylation
(91) A luciferase-based cytotoxicity assay was established using the luciferase transduced MM.1S-Luc cell line. In this assay, bioluminescence is only detected from living cells since luciferase released by dead cells is unable to function due to the lack of ATP in the medium. PBMCs from healthy donors were isolated and mixed with MM.1S-Luc cells in a ratio of 20 to 1. After 4 hours the bioluminescence was measured.
(92) With a selection of 4 unstimulated donor PBMC preparations, the in vitro cytotoxicity of J22.9-xi was determined. The cytotoxic potential varies slightly between PBMCs from different donors. Within 4 hours of incubation, cell lysis reached 18 to 35% at a concentration of 125 ng/ml J22.9-xi. Increasing the J22.9-xi concentration to 1 ug/ml increased cell lysis up to 56% (
(93) After deglycosylation of J22.9-xi (J22.9-xi-N-glycan) with PNGase F, the cytotoxic activity dropped to below 8%, whereas the binding of J22.9-xi-N-glycan to BCMA-positive MM.1S cells remained unaltered (
(94) J22.9-xi Reduces Tumor Burden in Xenografted Mice and Prolongs Survival
(95) We used NOD scid common gamma chain knock out (NSG) mice lacking functional B, T and NK cell populations. These mice, injected with 1*10.sup.7 MM.1S-Luc cells intravenously, develop hind limb paralysis within 6 weeks (
(96) After injection of 1×10.sup.7 MM.1S-Luc in the tail vein, the mice were divided randomly into 3 groups. The first group (n=2) received no treatment until the end of the experiment, whereas the second (n=5) and the third (n=6) group received twice weekly injections of 200 μg of an isotype control or the J22.9-xi antibody, respectively. The antibodies were administered for a period of 6 weeks intraperitoneally (i.p.) starting with the day of tumor cell injection. Tumor growth was monitored once a week using the IVIS Spectrum. Bioluminescence was measured 3 minutes after i.p. injection of luciferin.
(97) A similar course of tumor development was seen in both the untreated group and the group receiving the control antibody, whereas the group treated with J22.9-xi showed significantly less tumor burden, already beginning at the first measurement point at day six (
(98) Administration of 200 μg of an antibody to a mouse corresponds to approximately 10 mg/kg bodyweight. To test the efficacy of J22.9-xi at lower doses we divided MM.1S-Luc-xenografted mice into four groups. The first group (n=7) received 200 μg of the control antibody twice weekly, and groups 2, 3 (each n=3) and 4 (n=9) were injected with 2 μg, 20 μg or 200 μg twice a week, respectively. Injection and monitoring were performed as described above.
(99) Although tumors developed as expected in the control group mice, dramatically restricted tumor growth was observed in the groups receiving 20 μg or 200 μg of J22.9-xi (
(100) Growth of Established Tumors Arrests for 5 Weeks During J22.9-xi Treatment
(101) Therapeutic administration was mimicked by delaying the start of antibody treatment to 5 days after tumor cell injection. The xenografted mice were divided into 2 groups (n=6). The animals received 200 μg per injection of either the isotype control or J22.9-xi antibody twice a week. The first measurement was done at day 8 post cell injection. While there is no tumor-derived bioluminescence measurable to day 35 in the group receiving J22.9-xi (n=5), a steady increase in tumor load was seen in animals receiving the isotype control antibody (n=6) (
(102) Intensive Early Phase Treatment with J22.9-xi Prevents Tumor Growth for 7 Weeks
(103) In order to further assess the effect of treatment timing on tumor growth, different antibodies were administered for five consecutive days starting from the day of tumor cell injection. Subsequent to i.v. cell injection, the animals were divided randomly into 5 groups. Group 1 (n=5) was treated with 200 μg of the isotype control antibody per injection (i.p.), whereas group 2 (n=6) received 200 μg/injection of the J22.9-xi-N-glycan antibody. The mice from groups 3 (n=4), group 4 (n=5) and group 5 (n=5) obtained 200 μg, 20 μg and 2 μg per injection of the J22.9-xi antibody, respectively. Bioluminescence measurements began at day 9 post cell injection. Up to day 44, no tumor-derived bioluminescence was seen in any of the groups receiving the intact J22.9-xi antibody. Although the tumor growth in the animals treated with J22.9-xi-N-glycan is decelerated, the overall tumor load is not significantly different from those animals receiving the isotype control antibody (
(104) Humanisation of J22.9-xi
(105) The J22.9-xi antibody was humanized based on sequence alignment and the data obtained from the crystal structure. The sequences of the variable regions were aligned to their respective human homologs using IgBLAST (NCBI) or Clustal (EBI). Each proposed mutation was evaluated by visual inspection of the structure before alteration.
(106) Binding of Humanized Variants to BCMA Target
(107) Binding of the mutants to BCMA was tested using flow cytometry, ELISA and SPR. The affinity of the humanized antibodies was measured using surface plasmon resonance (ProteOn™ XPR36; Bio-Rad). The binding data show surprising results with respect to the specificity and affinity of the humanized antibody variants to the same epitope as tested for J22.9-xi binding. As shown in the table below, it was entirely surprising that the humanized antibodies as described herein exhibited comparable binding characteristics as the original chimeric antibody. The SPR data reveals that the affinities of the humanized variants are similar to those of the chimera and are sufficient to assume their clinical relevance in light of the data provided herein for the original chimeric antibody. A skilled person would not have expected that through the modification of the CDRs during humanization of the chimera that the binding characteristics would be maintained to such an extent.
(108) ELISA was carried out as described herein using BCMA-coated microtiter plates (1 microg/ml). As observed in
(109) Flow cytometry was also carried out using the humanized variants described herein and equivalent binding to both human and cynomlgous BCMA for all humanized variants tested was shown (refer
(110) SPR analysis was also conducted and affinities measured for humanized antibody variants. As can be observed in the table below (table 6), the affinities of the humanized variants (J22.9-H corresponds to humanized sequence SEQ ID No. 27; J22.9-FSY corresponds to humanized and PTM modified SEQ ID No. 28; J22.9-ISY corresponds to humanized and PTM modified SEQ ID No. 29).
(111) TABLE-US-00023 TABLE 6 SPR Data Affinity (SPR) Affinity (SPR) ELISA ELISA Flow Melting (human) (cynomolgous) Name (human) (cynomolgous) cytometry temperatures (n = 3) (n = 2) J22.9-xi +++ +++ +++ 86/94° C. 2.8 ± 0.7 × 2.7 × 10.sup.−9M 10.sup.−10M J22.9-H ++ + nd 86/94° C. 1.5 ± 0.3 × 2.0 × 10.sup.−7M 10.sup.−8M J22.9- +++ +++ +++ 87/94° C. 2.2 ± 0.3 × 2.0 × 10.sup.−8M FSY 10.sup.−9M J22.9- +++ +++ +++ 86/94° C. 2.0 ± 0.2 × 1.7 × 10.sup.−8M ISY 10.sup.−9M
(112) Amino Acid 54 in CDR2 of the J22.9 Heavy Chain:
(113) In order to remove a potential post-translational modification site in the humanized J22.9, residue D54 of the heavy chain CDR2 was mutated to asparagine (N), inadvertently creating a new potential modification site for N-linked glycosylation. The mutated heavy chain containing N54 migrated slower on SDS gels (
(114) The corresponding IgG, J22.9-FNY, nevertheless bound BCMA in FACS and ELISA, and was crystallized in complex with BCMA. Although not completely refined, the 2.7 Angstrom resolution structure shows clear electron density extending from the N54 side chain —consistent with a sugar modification of the residue. It is surprising that such a large extension of the side chain would not disrupt binding to BCMA and it could be expected from these observations that multiple and various amino acid substitutions would be tolerated at this position, potentially also derivatizations other than sugars.
(115) Methods
(116) Cell Lines and Culture
(117) The human multiple myeloma cell line MM.1S (Greenstein et al. (2003) Exp Hematol 31:271-282) was obtained from Prof. B. Dörken (MDC, Berlin, Germany). For in vivo monitoring of tumor cell growth, Luciferase and GFP were cloned into the pFU vector of the lentiviral vector system ViraPower (Invitrogen). Via GFP-expression of transduced cells, monoclonal cell lines were isolated using fluorescence-activated single cell sorting. Cell lines were cultured in RPMI-1640 medium without phenol red, containing 10% fetal calf serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin (all from PAA).
(118) The HEK293-6E cells, purchased from the National Research Council of Canada, were maintained in Freestyle F17 medium (Invitrogen) supplemented with 7.5 mM L-Glutamine (PAA), 0.1% Pluronic F-68 (Invitrogen), and 25 μg/ml G418 (Invitrogen). Cells were grown in Erlenmeyer flasks (Corning) at 110 rpm and 37° C. in a 5% CO2 atmosphere.
(119) Antibody Production and Purification
(120) To obtain a BCMA-binding antibody, standard hybridoma technique was used. 4 BL/6 wild type mice were immunized 6 times with incomplete Freund's adjuvant and 30 μg of the extracellular domain of human BCMA C-terminally fused to Glutathione S-transferase (GST). After cell fusion followed by a screening period the J22.9 hybridoma was shown to secrete an anti-BCMA antibody.
(121) Due to the instability of the hybridomas the variable regions of the light and heavy chain of hybridoma J22.9 were amplified and cloned upstream of the human kappa or the IgG1 constant domain genes, respectively. The chimeric J22.9-xi antibody was produced by transient cotransfection of 293-6E cells with a 1:2 DNA plasmid mixture encoding the light and heavy chains, respectively. In brief: 293-6E cells were resuspended to 1.7×10.sup.6 cells/ml in serum free Freestyle F17 medium and transfected using polyethyleneimine at a final concentration of 1 μg/ml culture. Two days after transfection, cells were fed with 100% of the transfection volume Freestyle F17 medium containing 1% tryptone N1 (Organo Technie). At day 7 cells were harvested by centrifugation and the filtered (0.45 μm) culture medium was passed over a 3.5 ml Protein A Sepharose column (Bio-Rad). The column was washed with 10 ml phosphate buffered saline (PBS) and antibody eluted by addition of 20 mM sodium acetate, 150 mM NaCl, pH 3.5. Fractions of 2 ml were collected directly into tubes containing 100 μl 1 M HEPES, pH 7.5 for neutralization. The final yield of full length IgG was approximately 40 mg/I culture.
(122) Since hybridoma J22.9 lost the capacity to produce/secrete the anti-BCMA antibody (
(123) The isotype control antibody composed of the J22.9-xi heavy chain and a random chimeric kappa light chain was produced in parallel with the J22.9-xi antibody. This antibody was shown by ELISA and flow cytometry to be unable to bind to BCMA.
(124) The N-linked oligosaccharide chains at Asn297 of the heavy chain of J22.9-xi were removed enzymatically using N-Glycosidase F (PNGase F) (NEB). 10 mg of J22.9-xi were incubated with 15,000 units PNGase F in 500 μl PBS (pH 7.4) for 36 hours at 37° C. followed by buffer exchange into sterile PBS.
(125) Determination of Binding and Blocking Capacities of J22.9-xi by Enzyme-Linked Immunosorbent Assays (ELISA)
(126) Microtiter plates were coated with 10 μg/ml of the extracellular domain of human BCMA. Coated BCMA was detected with serial dilution of J22.9-xi and the isotype control ranging from 1 to 1000 ng. Binding of J22.9-xi or isotype control antibody to the coated BCMA was detected with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch, 109-035-098, dilution 1:5,000).
(127) Microtiter plates were coated with 1 μg/ml of the extracellular domain of human or cynomolgous BCMA (hBCMA or cyBCMA, respectively). Coated BCMA was detected with serial dilution of J22.9-xi, J22.9-H, J22.9-ISY and J22.9-FSY ranging from 0.26 pM to 500 nM. Binding of antibodies to the coated BCMA was detected with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch, 109-035-098, dilution 1:5,000).
(128) For the blocking experiment, 1 mg/ml of human recombinant BAFF fused to a His-tag (Biomol) was applied after the antibodies and washing and detected using the mouse anti-His tag (AbD Serotec, AD1.1.10, dilution 1:5,000, HRP-conjugated) antibody. All ELISAs were developed using BD OptEIA reagents A and B (BD Bioscience) and measured with a microplate spectrophotometer (BioTek) at 450 nm and 570 nm.
(129) Flow Cytometry Analysis
(130) For cell surface antigen detection experiments, self-made antibodies (J22.9-xi, J22.9-H, J22.9-ISY, J22.9-FSY and the isotype control) and commercially available mouse anti-His tag (AbD Serotec, AD1.1.10, dilution 1:100, Alexa Fluor 488-conjugated) and goat anti-human IgG1 (Jackson ImmunoResearch, 109-116-098, dilution 1:400, PE-conjugated) antibodies and human recombinant BAFF fused to a His-tag (Biomol) were used. Experiments were performed on a FACSCalibur or a FACSCanto II flow cytometer (BD Bioscience). The data were analysed with Flowjo software version 7.6 (TreeStar Inc.).
(131) Generation of Fab and Fab:BCMA Complexes
(132) (Fab).sub.2 fragments were generated from full length J22.9-xi IgG by incubation with pepsin. J22.9-xi was passed over a PD-10 buffer exchange column into 50 mM sodium acetate, pH 3.5 and pepsin added at 30 μg per milligram J22.9-xi. Incubation at 37° C. for 2.5 hours was sufficient to completely digest the fragment crystallizable (Fc) region and pepsin was inactivated by exchange over a PD-10 column into PBS (pH 7.2). The reduction of the (Fab).sub.2 fragments to individual Fabs was accomplished in PBS by addition of 2-Mercaptoethylamine (50 mM) in the presence of 5 mM ethylenediaminetetraacetic acid (EDTA). After incubation for 90 minutes at 37° C., the reduced cysteines were blocked by alkylation with 500 μM iodoacetamide for 30 minutes followed by buffer exchange into fresh PBS. The Fab fragments were combined with 1.5 molar equivalents of purified BCMA and the complexes isolated by size exclusion chromatography on a Superdex 75 16/60 column. Fractions were analyzed on 4-12% SDS polyacrylamide gels and fractions containing both Fab and BCMA were pooled and concentrated for crystallization trials.
(133) Crystallization of Fab:BCMA Complexes
(134) Concentrated complexes were supplemented with 0.5 molar equivalents of pure BCMA to ensure saturation and were subjected to crystallization screening. Initial Fab:BCMA crystallization conditions were identified from commercial screens (Qiagen) in 96-well sitting drop format plates using a Gryphon pipetting robot (200 nl drops) and optimized in 24 well plates in hanging drops (2-3 ul). The complex was concentrated to 8 mg/ml and crystallized in 21% PEG 3350, 0.1 M BisTris pH 6.5 and 5 mM CuCl.sub.2 at 20° C. Crystals appeared after three days as clusters of thin plates and attained their final size (0.2-0.3 mm) within approximately 7 days. Clusters were separated and individual plates were flash frozen in liquid nitrogen in mother liquor with 20% glycerol as cryoprotectant. Complete diffraction data was collected from a single crystal at the BESSY synchrotron of the Helmoltz Zentrum Berlin. The structure was solved to a resolution of 1.9 angstroms by molecular replacement using the experimental phases from the structure of Efalizumab (3E09) as the search model. Data processing was performed with the ccp4 suite of programs, structure refinement was performed using Phenix (Adams P D, et al. (2010), Acta Cryst. D66: 213-221) and model building and assessment using Coot. (Emsley et al, Acta Crystallographica Section D—Biological Crystallography, 2010, 66:486-501) Images were made using PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
(135) In Vitro Cytotoxicity Assay
(136) In this assay the cytotoxic effect of J22.9-xi was determined by measuring the luminescence of the remaining living cells in a bioluminescence reader. In short: freshly obtained human filter buffy coats (FBC) were back-flushed by gravity with 160 ml elution buffer (PBS (pH 7.4) containing 5 mM Na.sub.2-EDTA and 2.5 [w/v] sucrose). Mononuclear cells were isolated from the eluted cells by Ficoll gradient centrifugation. Mononuclear cells from the interphases were taken and washed twice in elution buffer. After erythrocyte lysis, PBMCs were washed again, counted and adjusted by dilution in RPMI/10% FCS w/o phenol red to 1*10.sup.7 cells/ml. 5*10.sup.4 MM.1S-Luc cells in 50 μl RPMI were plated in microtiter plates. Ten minutes prior to the addition of 100 μl PBMCs, the MM.1S-Luc cells were incubated with J22.9-xi or the isotype control antibody serial dilutions in a sample volume of 200 μl. After addition of target cells, antibodies and effector cells, microtiter plates were centrifuged (300×g) for 2 minutes at room temperature (RT) and stored at 37° C. with 5% CO2. Control wells were treated with 1% Triton X instead of antibody for complete lysis. After 4 hours of incubation, 25 μl of PBS with luciferin (250 ng/ml) were applied to each well, and the bioluminescence of the living cells was measured in a bioluminescence reader (Tecan). The specific cytotoxicity was calculated according to the following formula:
100−[value(J22.9-xi)−value(total lysis)]/[value(isotype control)−value(total lysis)]*100.
(137) In Vivo Studies
(138) NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CSF2)2Ygy Tg(IL3)1Ygy Tg(KITLG)3YgyJGckRolyJ mice (NSG) from The Jackson Laboratory and CB17.Cg-Prkdcscid Lystbg/Crl mice from Charles River Deutschland (Sulzfeld, Germany) were used. Experiments were performed with mice between 8-14 weeks old. All animal studies were performed according to institutional and state guidelines, under specific pathogen-free conditions. In the experimental examples relating to treatment of established tumours and tumour treatment in the early phase of disease the CB17.Cg-Prkdcscid Lystbg/Crl mice were used. The phenotype of the two mice strains mentioned herein is very similar. The animals have no functional B-, T- and NK-cells. A slightly slower tumour growth was observed in the CB17.Cg mice, indicating an even more promising effect of the therapeutic antibody of the present invention.
(139) The xenograft model of multiple myeloma was induced by intravenous injection of 1*10.sup.7 MM.1S-Luc cells in the tail vein at day zero. In this model, untreated animals develop hind limb paralysis within 6 weeks. Occurrence of this symptom indicates the end point of the experiment.
(140) For the efficacy studies, the antibodies were administered intraperitoneally (i.p.) twice a week or on 5 consecutive days starting at day zero. The J22.9-xi antibody was given in doses of 2 μg, 20 μg or 200 μg per injection; for the isotype control antibody, 200 μg/injection was used. The bioluminescence of the MM.1S-Luc cells was measured after i.p. injection of 150 μg luciferin using the IVIS Spectrum (Caliper Life Sciences). Measurements were done weekly. At each timepoint, 3 untreated control mice were also administered luciferin. Total flux values of these animals are either subtracted from each measurement or shown in the graphs.
(141) To treat established tumors, antibody therapy was begun 5 days after injection of the MM.1S-Luc cells. 200 μg of the J22.9-xi or isotype control antibody was administered twice a week for a period of 6 weeks.
(142) Humanization of J22.9-xi
(143) The heavy and light chain variable region sequences (mouse) were aligned with those from the corresponding heavy and light chain subtype human sequences to determine which residue alterations were required to produce a fully humanized sequence variant. Using the crystal structure of the J22.9-xi:hBCMA complex, each modification was first assessed in silico to identify mutations that could potentially disrupt binding of the antibody to BCMA. Two complete J22.9 variable region genes for each chain were synthesized, one with the original mouse sequence and one with a completely humanized sequence (i.e. containing all of the necessary humanizing mutations) with two added restriction enzyme sites to divide the genes into three cassettes each. After flagging potentially problematic mutations, various combinations of the original mouse and fully humanized gene cassettes were produced and their corresponding IgGs were expressed, purified and subjected to FACS analysis with BCMA positive cells to assess binding. Flagged problematic residues were mutated individually using PCR to verify their effect on affinity to BCMA and the final optimized constructs were subsequently quantitatively assessed for binding to both human and cynomolgus BCMA via SPR.
(144) SPR
(145) SPR was performed on a ProteonXPR36 using phosphate buffered saline supplemented with 0.005% Tween-20 (PBST). Whole IgG at a concentration of 15 ug/ml was immobilized to a Proteon GLH sensor chip using standard amine chemistry according to the manufacturer's instructions. For binding experiments, human or cynomolgus BCMA in PBST was used as the mobile phase. Binding affinities (K.sub.d) were calculated from association (k.sub.on) and dissociation (k.sub.off) constants determined in parallel at multiple concentrations of BCMA (ranging from 0.4 to 800 nM for hBCMA and 2.7 nM to 1 μM for cynoBCMA) assuming a single-site binding model.
(146) Additionally, further experimentation shows that the preferred embodiments of the invention provide surprising and unexpected effects, thereby solving the problem of the invention in a non-obvious fashion.
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