CHANGES OF IGM FC AND J CHAIN AFFECTING THE SERUM IGM SERIE LIFE

专利摘要:
igm fc and j chain mutations affecting the igm serum half-life. this disclosure provides an igm antibody or igm-like antibody comprising variant j and / or heavy chain constant regions of variant igm that can impart increased serum half-life to the antibody.
公开号:BR112020017296A2
申请号:R112020017296-0
申请日:2019-03-01
公开日:2020-12-29
发明作者:Ramesh Baliga；Bruce Alan Keyt；Dean Ng
申请人:Igm Biosciences, Inc.；
IPC主号:

专利说明:

[001] [001] This application claims the benefit of US Provisional Patent Application Serial No. 62 / 637,186, filed on March 1, 2018, which is incorporated into this document by reference in its entirety. REFERENCE TO THE SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[002] [002] The contents of the sequence listing sent electronically in the ASCII text file (Name “09789-012WO1-Sequence-Listing; Size: 98,048 bytes; and Creation Date: February 28, 2019”) deposited with the request is incorporated in this document by reference in its entirety. FUNDAMENTALS
[003] [003] Antibodies and antibody-like molecules that can multimerize, such as IgA and IgM antibodies, have emerged as promising candidates for drugs in the fields of, for example, immuno-oncology and infectious diseases, allowing for greater specificity, greater avidity and the ability to bind to multiple liaison targets. See, for example, US Patents No. 9,951,134 and 9,938,347 and PCT Publications No. WO 2016/141303, WO 2016/154593, WO 2016/168758, WO 2017/059387, WO 2017 059380, WO 2018/017888, WO 2018/017763, WO 2018/017889 and WO 2018/017761, the contents of which are hereby incorporated by reference in their entirety.
[004] [004] The pharmacokinetics (PK) and pharmacodynamics (PD) of multivalent antibodies are, however, complex and depend on both the structure of the monoclonal antibody and the physiological system for which it is intended. In addition, different classes of antibodies are typically processed within a subject through different cellular and physiological systems. For example, the class of IgG antibodies has a serum half-life of 20 days, while the half-life of IgM and IgA antibodies is only 5–8 days. Brekke, OH., And I. Sandlie, Nature Reviews Drug Discovery 2: 52-62 (2003).
[005] [005] The antibody molecules in vivo can bind to a variety of receptors in various blood cells or in different tissues and organs. The binding to these receptors can influence the bioavailability and biodistribution of therapeutic antibodies and their ability to reach the targets of interest. For example, antibodies of the IgG isotype are known to have a long half-life in vivo due to the binding of the recycling neonatal Fc receptor (FcRn). However, IgA and IgM isotype antibodies do not bind to this key recycling receptor. IgM isotype antibodies are known to bind to the Fc µ receptor (FcμR), to the Fc α / µ receptor (FcαμR) and to the polymeric Ig receptor (pIgR). IgM antibodies with and without J chain bind to FcμR (Kubagawa, H., et al., Curr. Top. Microbiol. Immunol. 408: 25-45 (2017)), but J chain residues at least contribute to the binding of IgM antibodies to FcαμR (Ghumra, A., et al., Eur. J. Immunol. 39: 1147-1156 (2009)) and the binding of IgM to pIgR depends on the J chain (Braathen R., et al J. Immunol 178: 1589-1597 (2007)). PIgR is responsible for transporting IgM and IgA to the lumen of the intestinal, salivary and lacrimal glands (see, for example, Braathen, R., et al., J. Biol. Chem.
[006] [006] The J chain is a 15 kDa acid polypeptide, which is associated with pentameric IgM and dimeric IgA through disulfide bonds involving the penultimate 18 amino acid (tailpiece) tail cysteine residue (tp)
[007] [007] Despite the advances made in the design of multimeric antibodies, it is still necessary to be able to manipulate the pharmacokinetic and pharmacodynamic properties of these molecules. SUMMARY
[008] This disclosure provides an IgM antibody or IgM-like antibody with increased serum half-life, wherein the antibody includes five divalent or variant antibody binding units or fragments thereof and a variant J chain or functional fragment thereof. Each binding unit of the antibody provided includes two IgM heavy chain constant regions or multimerization fragments or subunits thereof, each associated with an antigen-binding domain or subunit thereof. In some respects, the variant J chain or functional fragment thereof includes one or more substitutions, deletions or insertions of unique amino acids in relation to a reference J chain identical to the variant J chain, except for one or more substitutions, deletions or insertions of amino acids unique. The supplied variant J chain can affect the serum half-life of the supplied IgM antibody or IgM-like antibody. For example, in certain aspects, the supplied IgM antibody or the IgM-like antibody exhibits an increased serum half-life after administration to an animal subject in relation to a reference IgM antibody or an IgM-like antibody that are identical, except for one or more substitutions, deletions, or insertions of unique amino acids in the variant J chain, and are administered in the same way to the same species of animals. In some respects, the variant J chain or functional fragment thereof includes one, two, three or four substitutions, deletions or insertions of amino acids unique to a reference J chain.
[009] [009] In some respects, the variant J chain or functional fragment thereof includes an amino acid substitution at the amino acid position corresponding to the wild type human J chain amino acid Y102 (SEQ ID NO: 2). For example, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by alanine (A), serine (S) or arginine (R). In certain respects, the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by alanine (A). In some respects, the J chain is a variant human J chain and includes the amino acid sequence SEQ ID NO: 3. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by serine (S). In some respects, the J chain is a variant human J chain and includes the amino acid sequence SEQ ID NO: 4.
[010] [010] In certain respects, an IgM antibody or IgM-like antibody that includes a variant J chain as provided above, the IgM heavy chain constant regions or multimerization fragments thereof may be constant regions of the variant IgM heavy chain, including a or more single amino acid substitutions, deletions or insertions with respect to a constant region of the reference IgM heavy chain identical to the constant regions of the variant IgM heavy chain, except for one or more single amino acid substitutions, deletions or insertions. According to these aspects, the regions of the variant IgM heavy chain may affect the serum half-life of the IgM antibody or IgM-like antibody, so that the supplied IgM antibody or IgM-like antibody exhibits an increased serum half-life additionally after administration to an animal subject with respect to a reference IgM antibody or an IgM-like antibody that are identical, except for one or more substitutions, deletions, or insertions of unique amino acids in the constant regions of the IgM heavy chain, and are administered in the same way to the same species of animals. In some ways, the additional increase in serum half-life is additive.
[011] [011] This disclosure further provides an IgM antibody or an IgM-like antibody with increased serum half-life, wherein the IgM antibody or the IgM-like antibody includes five or six divalent antibody binding units or their variants or fragments and wherein each binding unit includes two variants of IgM heavy chain constant regions or their multimerization fragments, each associated with an antigen binding domain or its subunit. According to these aspects, the regions of the variant IgM heavy chain or its multimerization fragments include one or more substitutions, deletions or insertions of unique amino acids in relation to a constant region of the reference IgM heavy chain identical to the regions contained in the IgM heavy chain variant, except for one or more single amino acid substitutions, deletions or insertions. In addition, according to these aspects, the regions of the variant IgM heavy chain can affect the serum half-life of the IgM antibody or IgM-like antibody. In certain respects, the supplied IgM antibody or the IgM-like antibody exhibits an increased serum half-life after administration to an animal subject compared to a reference IgM antibody or an IgM-like antibody that are identical, except for a or more single amino acid substitutions, deletions, or insertions in the constant regions of the IgM heavy chain, and are administered in the same way to the same species of animals. In certain respects, the regions of the variant IgM heavy chain include one, two, three or four unique amino acid substitutions, deletions or insertions in relation to the constant region of the reference IgM heavy chain.
[012] [012] In certain respects, the variant IgM heavy chain constant regions include an amino acid substitution at the amino acid position corresponding to amino acid R344 of the wild-type human IgM constant region SEQ ID NO: 12, for example, the amino acid corresponding to R344 of SEQ ID NO: 12 can be replaced by alanine (A). In certain respects, the variant IgM heavy chain constant regions are heavy chain constant regions
[013] [013] In an IgM antibody or IgM-like antibody, as provided in this document, the increased serum half-life may include an increased alpha half-life (t1 / 2α), an increased beta half-life (t1 / 2β), or an increase of t1 / 2α and an increase of t1 / 2β. Likewise, an IgM antibody or IgM-like antibody, as provided in this document, may also exhibit an increased peak plasma concentration (Cmax), an increased area under the curve (AUC), a modified clearance time or any combination of compared to the reference antibody.
[014] [014] In certain respects, regions of the IgM heavy chain or multimerization fragments or variants thereof of an IgM antibody or IgM-like antibody, as provided herein, each includes a Cµ4 domain and an IgM tail domain (tp) and can also include a Cµ3 domain, a Cµ2 domain, a Cµ1 domain or any combination thereof.
[015] [015] In certain respects, the antigen-binding domain of an IgM antibody or IgM-like antibody, as provided herein, may be a single chain Fv fragment (ScFv) or a single domain variable region (VHH). In certain respects, the subunit of the antigen-binding domain of an IgM antibody or IgM-like antibody, as provided herein, may be a variable region of the heavy chain (VH).
[016] [016] In certain respects, each binding unit of an IgM antibody or IgM-like antibody, as provided herein, may also include two light chain constant regions or fragments or variants thereof, each associated with a antigen or subunit thereof, for example, the antigen binding domain can be a scFv fragment or the subunit of the antigen binding domain can be a VL.
[017] [017] In certain respects, the J chain or functional fragment or variant thereof of an IgM antibody or IgM-like antibody, as provided herein, may further include one or more heterologous polypeptides, directly or indirectly, fused to the J chain or fragment functional or variant thereof. In certain aspects, the one or more heterologous polypeptides can be fused to the J chain or fragment thereof via a peptide linker. Exemplary linkers can include at least 5 amino acids, but no more than 25 amino acids, and can consist of GGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), GGGGSGGGGSGGGGS (SEQ ID NO: 27), GGGGGGGGGGGGGSG ID NO: 28), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 29). In certain aspects, the one or more heterologous polypeptides can be fused to the N-terminal of the J chain or fragment or variant thereof, the C-terminal of the J chain or fragment or variant thereof or fused to both the N-terminal and C-terminal of the J chain or fragment or variant thereof. Where two or more heterologous polypeptides are fused to the J chain, the heterologous polypeptides can be the same or different. In certain aspects, the at least one heterologous polypeptide can be a binding domain, for example, an antibody or antigen-binding fragment thereof, for example, a Fab fragment, a Fab 'fragment, an F (ab') fragment 2, an Fd fragment, an Fv fragment, a single chain Fv fragment (scFv), a disulfide-bound Fv fragment (sdFv) or any combination thereof. In some respects, the antigen-binding fragment is an scFv fragment. In certain respects, the at least one heterologous polypeptide can specifically bind to CD3ε. In certain aspects, the J chain can be a variant of the modified J chain of SEQ ID NO: 9 (V15J), for example, the amino acid sequence of SEQ ID NO: 10 (V15J-Y102A), the amino acid sequence of SEQ ID NO: 23 (V15J-T103A) or the amino acid sequence of SEQ ID NO: 24 (V15J-N49A).
[018] [018] This disclosure further provides a composition that includes the IgM antibody or IgM-like antibody, as provided herein, and a pharmaceutically acceptable carrier.
[019] [019] This disclosure further provides a J chain or a functional fragment thereof that includes one or more substitutions, deletions or insertions of unique amino acids in relation to a reference J chain that is identical, except for one or more substitutions, deletions or insertions of unique amino acids, where the variant J chain can affect the serum half-life of an IgM antibody or IgM-like antibody that includes the variant J chain. In some respects, the variant J chain includes the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 24, or any combination thereof.
[020] [020] This disclosure further provides an isolated polynucleotide including a nucleic acid encoding an IgM or IgM-like subunit polypeptide as provided herein, where the subunit polypeptide includes (a) a heavy chain constant region IgM or similar to IgM or a multimerization fragment thereof, (b) an antibody light chain, or (c) a J chain, a modified J chain or fragment or functional variant thereof, or (d) any combination thereof . In certain aspects, the subunit polypeptide includes an IgM or IgM-like heavy chain constant region or a multimerization fragment thereof, for example, the subunit polypeptide may include the amino acid sequence SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 34.
[021] [021] This disclosure further provides a method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies that include variant J chains. The method includes testing for pentameric IgM antibodies or pentameric IgM-like antibodies that include variant J chains or fragments thereof to increase the serum half-life in an animal subject to a reference pentameric IgM antibody or pentameric IgM-like antibody , where the variant J chains or fragments thereof include defined amino acid insertions, deletions or substitutions, and where the reference pentameric IgM antibody or pentameric IgM-like antibody includes a J chain or fragment thereof identical to the variant J chains, except for defined amino acid insertions, deletions or substitutions; and recovering the variant J chains or fragments thereof that confer increased serum half-life to the pentameric IgM antibodies or pentameric IgM-like antibodies relative to the reference pentameric IgM antibody or pentameric IgM-like antibody.
[022] [022] This disclosure further provides a method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies, including variant J chains. The method includes testing for pentameric IgM antibodies or pentameric IgM-like antibodies that include variant J chains or fragments thereof for their level of binding to the Fc alpha-mu receptor (FcαμR), to the polymeric Ig receptor (pIgR)
[023] [023] This disclosure further provides a method for identifying constant regions of the variant IgM heavy chain that can increase the serum half-life of an IgM antibody or IgM-like antibody that includes the constant regions of the variant IgM heavy chain. The method includes testing IgM antibodies or IgM-like antibodies that include constant regions of the IgM heavy chain variant to increase the serum half-life in an animal subject to a reference pentameric IgM antibody or pentameric IgM-like antibody, in that the regions of the variant IgM chain include defined insertions, deletions or substitutions of amino acids, and where the reference IgM antibody or IgM-like antibody includes regions of the IgM heavy chain that are identical to the regions of the variant IgM heavy chain, except those defined amino acid insertions, deletions or substitutions; and recovering those IgM antibodies or IgM-like antibodies including the IgM variant heavy chain constant regions that give increased serum half-life to the IgM antibodies or IgM-like antibodies including the IgM variant heavy chain constant regions to the reference IgM antibody or IgM-like antibody.
[024] [024] This disclosure further provides a method for identifying constant regions of the variant IgM heavy chain that can increase the serum half-life of an IgM antibody or IgM-like antibody including the regions of the variant IgM heavy chain. The method includes testing for IgM antibodies or IgM-like antibodies, including constant regions of the IgM heavy chain variant for their level of binding to the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcµR), to the polymeric Ig receptor ( pIgR), any combination of two of the receptors, or of the three receptors, where the regions contained in the variant IgM heavy chain include defined amino acid insertions, deletions or substitutions; and recovering those IgM antibodies or IgM-like antibodies, including variant IgM heavy chain constant regions that provide reduced FcαµR binding capacity, reduced FcµR binding capacity, reduced pIgR binding capacity, reduced binding capacity to any of the two receptors or reduced ability to bind to all three receptors, on IgM antibodies or IgM-like antibodies, including the IgM heavy chain constant regions relative to a reference IgM antibody or IgM-like antibody that includes constant regions on the chain IgM heavy chains identical to the constant regions of the variant IgM heavy chain, except for defined amino acid insertions, deletions or substitutions. This method may further include testing for recovered IgM antibodies or IgM-like antibodies that include constant regions of the variant IgM heavy chain to increase the serum half-life in an animal subject relative to a reference IgM antibody or IgM-like antibody, which includes variant IgM heavy chain constant regions identical to variant IgM heavy chain constant regions, except for defined amino acid insertions, deletions or substitutions. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES
[025] [025] Figures 1A-1B shows an alignment of the amino acid sequence of the human IgM heavy chain constant region (SEQ ID NO: 12) with that of the mouse (GenBank: CAC20701.1, SEQ ID NO: 16), cynomolgus monkey (GenBank: EHH62210.1, SEQ ID NO: 30), rhesus monkey (GenBank: AAD02420.1, SEQ ID NO: 17), chimpanzee (GenBank: PNI10622.1, SEQ ID NO: 18) and Sumatran orangutan (GenBank : PNJ04968.1, SEQ ID NO: 19). The amino acids corresponding to amino acids R345, E346, S401, E402 and E403 of SEQ ID NO: 12 are boxed.
[026] [026] Figure 2 is a table that summarizes the binding of human and mouse immunoglobulin receptors to bispecific anti-CD20 / anti-CD3 IGMs comprising several mutations in the J chain constant region and / or in the IgM heavy chain measured by ELISA . "WT" means anti-CD20 antibodies 1.5.3 with the IgM constant regions and V15J regions, as described in PCT Publication No. WO 2016/141303 before the introduction of alanine substitutions. The percentages indicate the extent of binding to the receptor relative to the initial "WT" antibody. The percentages indicate the level of receptor binding in relation to an IgM pentamer comprising constant regions of the wild type IgM heavy chain ("IgM", SEQ ID NO: 12) and a modified J chain comprising a fused wild-type human J chain in its N-terminal to a scFv that binds to CD3 ("VJ" or "V15J", SEQ ID NO: 9), which is designated as 100%.
[027] [027] Figures 3A-3E are graphs showing the attachment of several bispecific anti-CD20 / anti-CD3 IgMs with amino acid substitution mutations at a position on the J chain corresponding to amino acid Y102 of SEQ ID NO: 2, to pIgR . Each graph compares a single mutant with the 1.5.3 "wild type" 1.5.3VJ IgM anti-CD20 antibody (closed squares). Figure 3A: Y102A mutation (open squares); Figure 3B: Y102F mutation (closed triangles); Figure 3C: Y102T mutation (open triangles); 3D figure: Y102S mutation (open diamonds); Figure 3E: Y102R mutation (closed diamonds).
[028] [028] Figure 4 is a table that summarizes the pharmacokinetic data in mice for anti-CD3 monospecific IgMs and bispecific anti-CD20 / anti-CD3 IGMs comprising several mutations in the J chain constant region and / or in the IgM heavy chain in compared to controls. "WT IgM" means anti-CD20 1.5.3 antibodies with the IgM constant regions, as described in PCT Publication No. WO 2016/141303 prior to the introduction of alanine substitutions. "WT J" and "WT VJ" are regions of wild type J chains ("J") or anti-CD3 modified J chains "VJ", as described in PCT Publication No. WO 2016/141303 before the introduction of the substitutions of alanine. "VJH" means the J-chain modified with anti-CD3 "VJ" further comprising human serum albumin fused to the C-terminal (presented herein as SEQ ID NO: 11). "A" is the serum antibody concentration measured at t1 / 2Alpha; "B" is the serum antibody concentration measured at t1 / 2Beta; “T1 / 2Alfa” is the alpha half-life (in hours); “T1 / 2Beta” is the beta half-life measured in hours, “C0” is the serum antibody concentration in μg / ml measured at time zero; AUC0-inf ”is the area under the zero to infinite time curve, measured in μg / ml * h; and “MRT” is the mean serum residence time for the antibody measured in hours.
[029] [029] Figure 5 is a curve showing the effect of the Y102A mutation of the J chain alone or in combination with the S401A or E401A mutations of the IgM heavy chain on the general serum half-life of a bispecific IgM anti-CD20 / anti- CD3 compared to an IgG antibody comprising the same anti-CD20 binding regions VH and VL (153 IgG). 153 IgG: open circles; IgM 1.5.3
[030] [030] Figure 6 is a table comparing pharmacokinetic parameters of IgM 1.5.3 V15J and IgM 1.5.3 V15J-N49A.
[031] [031] It is to be noted that the term "one" or "an" entity refers to one or more of that entity; for example, "a binding molecule," is understood to represent one or more binding molecules. As such, the terms "one" (or "one"), "one or more" and "at least one" can be used interchangeably in this document.
[032] [032] In addition, "and / or", where used in this document, should be taken as the specific disclosure for each of the two specified characteristics or components with or without the other. Thus, the term "and / or" as used in a sentence such as "A and / or B" in this document is intended to include "A and B", "A or B", "A" (by itself) and "B" (by itself). Likewise, the term "and / or" as used in a sentence such as "A, B and / or C" is intended to cover each of the following modalities: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (by itself); B (by itself); and C (alone).
[033] [033] Unless otherwise defined, technical and scientific terms used in this document have the same meaning as commonly understood by someone ordinarily versed in the technique to which this disclosure is reported. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those versed in the technique with a general dictionary of many of the terms used in this disclosure.
[034] [034] Units, prefixes and symbols are denoted in their accepted form by the Système International de Unites (International System of Units - SI). Numeric ranges include the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino orientation for carboxy. The headings provided in this document are not limitations on the various aspects or aspects of the disclosure, which can be obtained by reference in the specification as a whole. Consequently, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
[035] [035] As used in this document, the term "polypeptide" is intended to encompass a "polypeptide" in the singular as well as in the plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other term used to refer to a chain or several chains of two or more amino acids, are included within the definition of "polypeptide", and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence. It can be generated in any way, including by chemical synthesis.
[036] [036] A polypeptide as disclosed in this document can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have that structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure, but can instead adopt a large number of different conformations and are referred to as unfolded.
[037] [037] An "isolated" polypeptide or fragment, variant or derivative thereof, means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Polypeptides produced recombinantly and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides that have been separated, fractionated or partially or substantially purified by any suitable technique.
[038] [038] As used herein, the term "a non-naturally occurring polypeptide" or any grammatical variants thereof is a conditional definition that explicitly excludes, but only excludes, the forms of the polypeptide that are, or can be, determined or interpreted by a judge, or an administrative or judicial body, to "occur naturally".
[039] [039] Other polypeptides disclosed in this document are fragments,
[040] [040] A "conservative amino acid substitution" is one in which an amino acid is replaced by another amino acid with a similar side chain.
[041] [041] The term "polynucleotide" is intended to encompass a nucleic acid in the singular, as well as nucleic acids in the plural, and refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA), cDNA or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, such as that found in peptide nucleic acids (PNA)). The terms "nucleic acid" or "nucleic acid sequence" refer to any one or more nucleic acid segments, for example, fragments of DNA or RNA, present in a polynucleotide.
[042] [042] An "isolated" nucleic acid or polynucleotide means any form of nucleic acid or polynucleotide that is separated from its native environment.
[043] [043] As used in this document, the term "a non-naturally occurring polypeptide" or any grammatical variants thereof is a conditional definition that explicitly excludes, but only excludes, forms of the nucleic acid or polypeptide that are, or can be, determined or interpreted by a judge, or an administrative or judicial body, as "naturally occurring".
[044] [044] As used in this document, a "coding region" is a portion of the nucleic acid that consists of codons translated into amino acids.
[045] [045] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide can normally include a promoter and / or other transcription or translation control elements operatively associated with one or more coding regions. An operable association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences in such a way as to place the expression of the gene product under the influence or control of the regulatory sequences. Two fragments of DNA (such as a polypeptide coding region and a promoter associated with it) are "operationally associated" if induction of the promoter function results in the transcription of the mRNA that encodes the desired gene product and if the nature of the link between the two DNA fragments do not interfere with the ability of regulatory expression sequences to direct expression of the gene product or interfere with the DNA model's ability to be transcribed. Thus, a promoter region would be operationally associated with a nucleic acid that encodes a polypeptide, if the promoter were able to transcribe that nucleic acid. The promoter can be a cell-specific promoter that directs substantial DNA transcription into predetermined cells. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors and transcription termination signals, can be operationally associated with the polynucleotide to direct cell-specific transcription.
[046] [046] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, cytomegalovirus promoter and enhancer segments (the first immediate promoter, in conjunction with intron-A), simian virus 40 (the first promoter) and retroviruses (like Rous's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone and rabbit ß-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Suitable additional transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (for example, interferon- or interleukin-inducible promoters).
[047] [047] Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons and elements derived from picornavirus (particularly an internal ribosome entry site, or IRES, also referred to as an ISCED sequence).
[048] [048] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), carrier RNA or ribosomal RNA.
[049] [049] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide, as disclosed in this document. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein, once the export of the growing protein chain through the rough endoplasmic reticulum has started. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the "full length" or full length polypeptide to produce a secreted form or "mature" of the polypeptide. In certain embodiments, the native signal peptide, for example, an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that maintains the ability to direct the secretion of the polypeptide that is operationally associated with it.
[050] [050] As used in this document, the term "binding molecule" refers in its broadest sense to a molecule that specifically binds to a receptor, for example, an epitope or an antigenic determinant. As further described in this document, a binding molecule can comprise one of the more "antigen-binding domains" described in this document. A non-limiting example of a binding molecule is an antibody or antibody-like molecule, as described in detail in this document which retains specific antigen binding. In certain aspects, a "binding molecule" comprises an antibody or antibody-like molecule, as described in detail in this document.
[051] [051] As used in this document, the terms "binding domain" or "antigen binding domain" (can be used interchangeably) refer to a region of a binding molecule, for example, an antibody or molecule antibody-like, which is necessary and sufficient to specifically bind to an epitope. For example, an "Fv", for example, a variable heavy chain and an antibody variable light chain, as two separate polypeptide subunits or as a single chain, is considered a "binding domain". Other antigen-binding domains include, without limitation, the variable heavy chain (VHH) of an antibody derived from a species of camelid or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold structure. A "binding molecule" or "antibody", as described in this document, can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more "antigen binding domains" .
[052] [052] The terms "antibody" and "immunoglobulin" can be used interchangeably in this document. An antibody (or a fragment, variant or derivative thereof, as disclosed herein) includes at least the variable domain of a heavy chain (for camelid species) or at least the variable domains of a heavy chain and a light chain. The basic structures of immunoglobulin in vertebrate systems are relatively well understood.
[053] [053] The term "immunoglobulin" comprises several broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (, , , , ) with some subclasses between them (for example, γ1- γ4 or 1 -2)). It is the nature of this chain that determines the "isotype" of the antibody such as IgG, IgM, IgA IgG or IgE, respectively. Immunoglobulin subclasses (subtypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. they are well characterized and are known to confer functional specialization. The modified versions of each of these immunoglobulins are easily discernible to the person skilled in the art, in view of the disclosure of the moment and, accordingly, are within the scope of this disclosure.
[054] [054] Light chains are classified as kappa or lambda (, ). Each heavy chain class can be linked to a kappa or lambda light chain. In general, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked together by covalent disulfide bonds or non-covalent bonds, when immunoglobulins are expressed, for example , by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the fork ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, for example, IgG antibodies, includes two subunits of the heavy chain and two subunits of the light chain connected covalently via disulfide bonds to form a "Y" structure, also referred to herein as an "H2L2 structure "or a" connecting unit ".
[055] [055] The term "binding unit" is used throughout this document to refer to the portion of a binding molecule, for example, an antibody, antibody-like molecule, antigen-binding fragment thereof or multimerization fragment thereof, which corresponds to a standard "H2L2" immunoglobulin structure, that is, two heavy chains or fragments thereof and two light chains or fragments thereof. In certain aspects, for example, where the binding molecule is a bivalent IgG antibody or antigen binding fragment, the terms "binding molecule" and "binding unit" are equivalent.
[056] [056] As used herein, an "IgM-like antibody" refers to a variant antibody that still retains the ability to form hexamers or, in association with the J chain, to form pentamers. An IgM-like antibody typically includes at least the Cµ4-tp domains of the IgM constant region, but can include domains of the heavy chain constant region of other antibody isotypes, for example, IgG, of the same species or of a different species. An IgM-like antibody can also be an antibody fragment in which one or more constant regions are deleted, provided that the IgM-like antibody is capable of forming hexamers and / or pentamers. Thus, an IgM-like antibody can be a hybrid IgM / IgG antibody or it can be a multimerization fragment of an IgM antibody.
[057] [057] The terms "valence", "bivalent", "multivalent" and grammatical equivalents, refer to the number of antigen-binding domains in a given binding molecule, for example, antibody or antibody-like molecule or in an connection unit. As such, the terms "bivalent", "tetravalent" and "hexavalent" in reference to a particular binding molecule, for example, an IgM antibody, IgM-like antibody or multimerization fragment thereof, denote the presence of two domains of antigen binding, four antigen binding domains and six antigen binding domains, respectively. A typical IgM antibody or IgM-like antibody, where each binding unit is bivalent, can have 10 or 12 valences. A bivalent or multivalent binding molecule, for example, antibody or antibody-like molecule, can be monospecific, that is, all antigen-binding domains are the same or can be bispecific or multispecific, for example, where two or more antigen-binding domains are different, for example, they bind to different epitopes on the same antigen or bind to completely different antigens.
[058] [058] The term "epitope" includes any molecular determinant capable of specific binding to an antigen-binding domain of an antibody or antibody-like molecule. In certain respects, an epitope may include chemically active surface clusters of molecules, such as amino acids, sugar, phosphoryl or sulfonyl side chains and, in certain respects, may have specific three-dimensional structural characteristics and / or charge characteristics. An epitope is a region of a target that is linked by an antigen-binding domain of an antibody.
[059] [059] The term "target" is used in the broadest sense to include substances that can be linked by a binding molecule, for example, antibody or antibody-like molecule. A target can be, for example, a polypeptide, nucleic acid, carbohydrate, lipid or other molecule. In addition, a "target" can, for example, be a cell, an organ or an organism comprising a linked epitope that can be linked by a binding molecule, for example, antibody or antibody-like molecule.
[060] [060] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of the variable light (VL) and heavy (VH) portions determine antigen recognition and specificity. On the other hand, the light chain (CL) and heavy chain domains (for example, CH1, CH2, CH3 or CH4) confer biological properties, such as secretion, transplacental mobility, binding to the Fc receptor, complement binding and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding or amino-terminal site of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 (or CH4 in the case of lgM) and CL regions actually comprise the carboxy-terminal of the heavy and light chains, respectively.
[061] [061] A "full length IgM heavy chain antibody" is a polypeptide that includes, towards N-terminal to C-terminal, a heavy chain variable domain (VH) antibody, a constant chain 1 domain antibody heavy chain (CM1 or Cμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), an antibody heavy chain constant domain 3 (CM3 or Cμ3) and an antibody heavy chain constant domain 4 (CM4 or Cμ4) that can include a tail.
[062] [062] As indicated above, the variable region (s) allows a binding molecule, for example, antibody or antibody-like molecule, to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and the VH domain, or subset of the complementarity determining regions (CDRs), of a binding molecule, for example, an antibody or antibody-like molecule, combine to form the antigen binding domain. . More specifically, an antigen-binding domain is defined by three CDRs on each of the VH and VL chains. Certain antibodies form larger structures. For example, IgA can form a molecule that includes two H2L2 binding units and a J chain covalently connected via disulfide bonds, which can be further associated with a secretory component, and IgM can form a pentameric molecule. or hexameric which includes five or six H2L2 binding units and, optionally, a J chain covalently connected via disulfide bonds.
[063] [063] The six "complementarity determining regions" or "CDRs" present in an antibody antigen binding region are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain, as the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the antigen-binding domain, referred to as "framework" regions, show less intermolecular variability. Framework regions largely adopt a folha-sheet conformation and CDRs form handles that connect, and, in some cases, form part of the--sheet structure. Thus, the framework regions act to form a scaffolding structure that provides the positioning of the CDRs in the correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent attachment of an antibody to its cognate epitope. The amino acids that make up the CDRs and framework regions, respectively, can be easily identified for any variable region of the heavy or light chain by a person skilled in the art in an ordinary way, since they have been defined in several different ways (see, " Sequences of Proteins of Immunological Interest, "Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987), which are incorporated into this document by reference in their entirety).
[064] [064] In the case where there are two or more definitions of a term that is used and / or accepted in the art, the definition of the term as used in this document is intended to include all of these meanings, unless explicitly stated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combination sites found within the variable region of light and heavy chain polypeptides. These particular regions have been described, for example, by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference. The definitions of Kabat and Chothia include overlap or subsets of amino acids when compared to each other. However, the application of both definitions (or other definitions known to those skilled in the art) to refer to a CDR of an antibody or a variant thereof must be within the scope of the term as defined and used in this document, unless otherwise indicated. otherwise. The appropriate amino acid residues that comprise CDRs, as defined by each of the references cited above, are shown below in Table 1 as a comparison. The exact amino acid numbers that span a specific CDR will vary depending on the sequence and size of the CDR. Those skilled in the art routinely determine which amino acids comprise a specific CDR, given the sequence of amino acids in the variable region of the antibody.
[065] [065] The variable domains of antibodies can also be analyzed, for example, using the IMGT information system (imgt_ponto_cines_ponto_fr /) (IMGT® / V-Quest) to identify variable region segments, including CDRs.
[066] [066] Kabat et al. they also defined a numbering system for variable domain sequences that is applicable to any antibody. A person skilled in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system presented by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless the use of the Kabat numbering system is explicitly observed, however, consecutive numbering is used for all amino acid sequences in this disclosure.
[067] [067] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, β-2 Microglobulins, Major Histocompatibility Antigens, Thy- 1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, α-2 Macroglobulins, and Other Related Proteins, ”US Dept. of Health and Human Services (1991). IgM constant regions can be numbered sequentially (ie amino acid # 1 starting with the first amino acid in the constant region or using the Kabat numbering scheme. A comparison of the human IgM constant region numbering sequentially (presented in this document as SEQ ID NO: 12) and the Kabat system is defined below The underlined amino acid residues are not counted in the Kabat system: Sequential numbering key (SEQ ID NO: 12) / KABAT for IgM heavy chain 1/127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF
[068] [068] Binding molecules, for example, antibodies, antibody-like molecules, antigen-binding fragments, variants or derivatives thereof and / or multimerization fragments thereof include, but are not limited to, polyclonal, monoclonal, human antibodies , humanized or chimeric, single chain antibodies, epitope-binding fragments, eg, Fab, Fab 'and F (ab') 2, Fd, Fvs, Single chain Fvs (scFv), single chain antibodies, Fvs bound to disulfide (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in US patent 5,892,019.
[069] [069] By "specifically binding", it is generally understood that a binding molecule, for example, an antibody or fragment, variant or derivative thereof, binds to an epitope via its antigen binding domain, and that binding implies some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule, for example, antibody or antibody-like molecule, is said to "specifically bind" to an epitope when it binds to that epitope, through its antigen-binding domain more easily than that would bind to a random, unrelated epitope. The term "specificity" is used in this document to describe the relative affinity by which a particular binding molecule binds to a particular epitope.
[070] [070] It can be said that a binding molecule, for example, an antibody or fragment, variant or derivative thereof disclosed in this document, binds to a target antigen with a dissociation rate (k (off)) less than or equal at 5 X 10-2 sec -1, 10-2 sec -1, 5 X 10-3 sec -1, 10-3 sec -1, 5 X 10-4 sec -1, 10-4 sec -1, 5 X 10-5 sec-1, or 10-5 sec-1 5 X 10-6 sec-1, 10-6 sec-1, 5 X 10-7 sec-1 or 10-7 sec-1.
[071] [071] It can be said that a binding molecule, for example, an antibody or fragment, variant or derivative of binding to the antigen disclosed in this document, binds to a target antigen with a higher association rate (k (on)) or equal to 103 M-1 sec-1, 5 X 103 M-1 sec-1, 104 M-1 sec-1, 5 X 104 M-1 sec-1, 105 M-1 sec-1, 5 X 105 M-1 sec-1, 106 M-1 sec-1, or 5 X 106 M-1 sec-1 or 107 M-1 sec-1.
[072] [072] A binding molecule, for example, an antibody or fragment, variant or derivative thereof, is said to competitively inhibit the binding of a reference antibody or antigen binding fragment to a given epitope if it preferably binds to that epitope in that it blocks, to some extent, the binding of the reference antibody or antigen-binding fragment to the epitope.
[073] [073] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, for example, of an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) on pages 27-28. As used in this document, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, for example, Harlow on pages 29-34. Avidity is related to the affinity of individual antigen-binding domains in the population with specific epitopes, and also the valences of immunoglobulins, and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, would be highly avid. An interaction between a bivalent monoclonal antibody and a receptor present in high density on the cell surface would also be highly avid.
[074] [074] Binding molecules, for example, antibodies or fragments, variants or derivatives thereof, as disclosed in this document, can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, for example, an antibody or fragment, variant or derivative thereof, specific for an antigen, to react with a second antigen; a measure of the relationship between two different antigenic substances. Thus, a binding molecule is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope usually contains many of the same complementary structural features as the induction epitope and, in some cases, can actually adapt better than the original.
[075] [075] A binding molecule, for example, an antibody or fragment, variant or derivative thereof, can also be described or specified in terms of its affinity for binding to an antigen. For example, a binding molecule can bind to an antigen with a dissociation or KD constant of no more than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10 -4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10-10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.
[076] [076] "Antigen-binding antibody fragments", including single chain antibodies or other antigen-binding domains, may exist alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3 domains or CH4, J chain or secretory component. Also included are antigen-binding fragments that can include any combination of variable region (s) with one or more of a hinge region, CH1, CH2, CH3 or CH4 domains, a J chain or a secretory component. The binding molecules, for example, antibodies or antigen binding fragments thereof, can be of any animal origin, including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another modality, the variable region may be the source of the origin (for example, of sharks). As used herein, "human" antibodies that have antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic to one or more human immunoglobulins and that in some cases express endogenous immunoglobulins and in some cases do not, as described below and, for example, in US Pat. No. 5,939,598 by Kucherlapati et al. According to aspects of the present disclosure, an IgM or IgM antibody as provided herein may include an antigen binding fragment of an antibody, for example, an scFv fragment, provided that the IgM or IgM antibody is capable of forming a multimer, for example, a hexamer or a pentamer.
[077] [077] As used herein, the term "heavy chain subunit" includes amino acid sequences derived from an immunoglobulin heavy chain, a binding molecule, for example, an antibody or antibody-like molecule comprising a heavy chain subunit. include at least one of: a VH domain, a CH1 domain, a hinge domain (for example, upper, middle and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain or a variant or fragment of the same. For example, a binding molecule, for example, an antibody, an antibody-like molecule or fragment, variant or derivative thereof, may include, without limitation, in addition to a VH domain: a CH1 domain; a CH1 domain, a hinge and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain and a CH3 domain. In certain aspects, a binding molecule, for example, an antibody, antibody-like molecule or fragment, variant or derivative thereof, may include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain and a J chain. In addition, a binding molecule, for example, antibody or antibody-like molecule, for use in the disclosure may not have certain portions of constant region, for example, all or part of a CH2 domain. It will be understood by one skilled in the art that these domains (e.g., the subunit of the heavy chain) can be modified so that they vary in the amino acid sequence of the original immunoglobulin molecule. According to aspects of the present disclosure, an IgM or IgM antibody as provided herein comprises sufficient portions of an IgM heavy chain constant region to allow the IgM or IgM-like antibody to form a multimer, for example, a hexamer or a pentamer .
[078] [078] As used in this document, the term "light chain subunit" includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit includes at least one VL and can also include a CL domain (for example, Cκ or Cλ).
[079] [079] Binding molecules, for example, antibodies, antibody-like molecules, or antigen-binding fragments, variants or derivatives thereof, or multimerization fragments thereof, can be described or specified in terms of epitope (s) or portion (s) of an antigen that they specifically recognize or bind to. The portion of a target antigen that interacts specifically with the antigen-binding domain of an antibody is an "epitope" or an "antigenic determinant". A target antigen can comprise a single epitope or at least two epitopes and can include any number of epitopes, depending on the size, conformation and type of antigen.
[080] [080] As previously indicated, the subunit structures and the three-dimensional configuration of the regions constant to various classes of immunoglobulins are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CH1 domain" includes the first domain of the constant region (plus amino terminal) of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of a typical IgG heavy chain molecule.
[081] [081] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends, for example, from amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360 , Kabat numbering system and amino acids 231-340, EU numbering system; see Kabat EA et al., op cit.The CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 amino acids Certain classes of immunoglobulins, for example, IgM, further include a CH4 region.
[082] [082] As used in this document, the term "hinge region" includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain in IgG, IgA, and IgD heavy chains. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.
[083] [083] As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The cysteine amino acid comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
[084] [084] As used in this document, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified) it is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (for example, mouse or primate) and the constant region is human.
[085] [085] The terms "multispecific antibody" "bispecific antibody" refer to an antibody or antibody-like molecule that has binding domains for two or more different epitopes within a single antibody molecule.
[086] [086] As used in this document, the term "engineered antibody"
[087] [087] As used in this document, the term "engineered" includes manipulation of nucleic acid or polypeptide molecules by synthetic means (for example, by recombinant techniques, peptide synthesis in vitro, by enzymatic or chemical coupling of peptides or some combination of these technical).
[088] [088] As used in this document, the terms "linked", "fused" or "fusion" or other grammatical equivalents can be used interchangeably. These terms refer to the joining of two or more elements or components, by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more open polynucleotide reading frames (ORFs) to form a larger continuous ORF, in a way that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (whose segments are not normally so united in nature). Although the reading frame is thus made continuous by all fused segments, the segments can be physically or spatially separated, for example, by an in-frame linker sequence. For example, polynucleotides that encode CDRs from an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide that encodes at least one immunoglobulin framework region or additional CDR regions, provided that the "fused CDRs" "are co-translated as part of a continuous polypeptide.
[089] [089] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino terminal sense for carboxyl in which amino acids neighboring each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino-terminal" or "N-terminal" for another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain. Likewise, a portion of a polypeptide that is "carboxy terminal" or "C-terminal" for another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain. For example, in a typical antibody, the variable domain is "N-terminal" for the constant region and the constant region is "C-terminal" for the variable domain.
[090] [090] The term "expression", as used in this document, refers to a process by which a gene produces a biochemist, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell, including, without limitation, knockdown of the gene, as well as transient and stable expression. It includes, without limitation, the transcription of the gene into RNA, for example, messenger RNA (mRNA) and the translation of that mRNA into the polypeptide (s). If the final desired product is a biochemical product, the term includes the creation of that biochemical product and any precursors. The expression of a gene produces a "gene product". As used in this document, a gene product can be either a nucleic acid, for example, a messenger RNA produced by the transcription of a gene or a polypeptide that is translated from a transcript. The gene products described in this document further include nucleic acids with post-transcriptional modifications, for example, polyadenylation, or polypeptides with post-translational modifications, for example, methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage and similar.
[091] [091] Terms such as "treating" or "treating" or "to treat" or "relieving" or "relieving" refer to therapeutic measures that cure, reduce, decrease the symptoms of, and / or interrupt or reduce the progression of an existing diagnosed pathological condition or disorder. Terms like "prevent", "prevention", "avoid", "impediment" and the like refer to prophylactic or preventive measures that prevent the development of an undiagnosed target condition or pathological disorder. Thus, "those in need of treatment" may include those who already have the disorder; those most likely to have the disorder; and those in whom the disease must be prevented.
[092] [092] As used in this document, the terms "serum half-life" or "plasma half-life" refer to the time it takes (for example, in minutes, hours or days) after administration for serum or plasma concentration of a drug, for example, a binding molecule, such as an antibody, antibody-like molecule or fragment thereof, as described in this document, to be reduced by 50%. Two half-lives can be described: the half-life
[093] [093] As used in this document, the term "area under the plasma drug concentration-time curve" or "AUC" reflects the body's actual exposure to the drug after administration of a dose of the drug and is expressed in mg * h / L. This area under the curve is measured from time 0 (t0) to infinity (∞) and depends on the rate of elimination of the drug from the body and the dose administered.
[094] [094] As used in this document, the term "average length of stay" or "MRT" refers to the average length of time that the drug remains in the body.
[095] [095] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammal subject, for which diagnosis, prognosis or therapy is desired. Mammals include humans, domestic animals, farm and zoo animals, sports or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears and so on.
[096] [096] As used in this document, phrases such as "a subject that would benefit from therapy" and "an animal that needs treatment" refer to a subset of subjects, among all potential subjects, that would benefit from administration of a particular therapeutic agent, for example, a binding molecule, such as an antibody, comprising one or more antigen-binding domains. Such binding molecules, for example, antibodies, can be used, for example, for diagnostic procedures and / or for treating or preventing a disease.
[097] [097] IgM is the first immunoglobulin produced by B cells in response to a stimulus from an antigen and is naturally present at about 1.5 mg / ml in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and therefore includes five or six binding units. An IgM binding unit typically includes two light chains and two heavy chains. Although an IgG heavy chain constant region contains three heavy chain constant domains (CH1, CH2 and CH3), the IgM heavy constant region (µ) additionally contains a fourth constant domain (CH4) and includes a C-terminal “tail” .
[098] [098] There are other forms of the human IgM constant region with small sequence variations, including, without limitation, GenBank Access Numbers.
[099] [099] Each IgM heavy chain constant region can be associated with an antigen binding domain, for example, a scFv or VHH, or a subunit of an antigen binding domain, for example, a VH region.
[0100] [0100] Five IgM binding units can form a complex with an additional small polypeptide chain (the J chain) to form a pentameric IgM antibody or IgM-like antibody. The precursor form of the human J chain, SEQ ID NO: 1, is shown below. The signal peptide (underlined) extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 1, and the mature human J chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 1.
[0101] [0101] Without the J chain, an IgM antibody or IgM-like antibody usually assembles in a hexamer, comprising up to twelve antigen-binding domains. With a J chain, an IgM antibody or IgM-like antibody generally assembles into a pentamer, comprising up to ten antigen-binding domains, or more if the J chain is a modified J chain comprising heterologous polypeptides that comprise domain (s) ) additional antigen binding (s). The assembly of five or six IgM binding units in an pentameric or hexameric IgM antibody or IgM-like antibody is thought to involve the Cµ4 and tail domains. See, for example, Braathen, R., et al., J. Biol. Chem.
[0102] [0102] In certain respects, the disclosure provides an IgM or Pentameric IgM-like antibody comprising five divalent binding units, wherein each binding unit includes two constant regions of the IgM heavy chain or multimerization fragments or subunits thereof, each associated to an antigen-binding domain or subunit thereof. In certain respects, the two regions on the IgM heavy chain are constant regions on the human heavy chain.
[0103] [0103] Where the IgM or IgM-like antibody provided herein is pentameric, the IgM or IgM-like antibody typically further comprises a J chain, or functional fragment or variant thereof. In certain respects, the J chain is a modified J chain or a variant thereof which further comprises one or more heterologous moieties attached to it, as described elsewhere in this document. In certain respects, the J chain can be mutated to affect, for example, improving the serum half-life of the IgM or IgM-like antibody provided in this document, as discussed elsewhere in this document.
[0104] [0104] An IgM heavy chain constant region may include one or more of a Cµ1 domain or fragment or variant thereof, a Cµ2 domain or fragment or variant thereof, a Cµ3 domain or fragment or variant thereof and / or a Cµ4 domain or fragment or variant thereof, provided that the constant region can serve a desired function in the IgM or IgM-like antibody, for example, to associate with the second IgM constant region to form an antigen-binding domain and / or to associate with other linking units (and in the case of a pentamer, a J chain) to form a hexamer or a pentamer. In certain respects, the two IgM heavy chain regions or fragments or variants thereof within an individual binding unit each comprise a Cµ4 domain or fragment or variant thereof, a tail (tp) or fragment or variant thereof, or a combination of a Cµ4 domain and a TP or fragment or variant thereof. In certain aspects, the two IgM heavy chain regions or fragments or variants thereof within an individual binding unit further comprise a Cµ3 domain or fragment or variant thereof, a Cµ2 domain or fragment or variant thereof, a Cµ1 domain or fragment or variant of this, or any combination of these.
[0105] [0105] In certain respects, the J chain of an IgM or IgM pentameric antibody, as provided herein, can be modified, for example, by introducing a heterologous fraction, or two or more heterologous fractions, for example, polypeptides , without interfering with the ability of the IgM antibody or similar IgM to assemble and bind to its binding target (s). See US Patent No. 9,951,134, PCT Publication No. WO 2017/059387 and PCT Publication No. WO 2017/059380, each of which is incorporated herein by reference in its entirety. Accordingly, IgM or IgM-like antibodies, as provided herein, including multispecific IgM or IgM antibodies, as described elsewhere in this document, may comprise a modified J chain or functional fragment or variant thereof comprising a heterologous fraction, for example , a heterologous polypeptide, introduced into the J chain or fragment or variant thereof. In certain aspects, the heterologous fraction may be a peptide or polypeptide sequence fused in structure to the J chain or chemically conjugated to the J chain or fragment or variant thereof, where the heterologous polypeptide is directly or indirectly fused to the variant J chain or functional fragment thereof . In certain aspects, the heterologous polypeptide is fused to the J chain or functional fragment of this pathway with a peptide linker, for example, a peptide linker consisting of at least 5 amino acids, but not more than 25 amino acids. In certain respects, the peptide linker consists of GGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), GGGGSGGGGSGGGGS (SEQ ID NO: 27), GGGGSGGGGSGGGGSGGG (SEQ IDGGG, SEG IDGG NO: 29). In certain aspects, the heterologous moiety may be a chemical moiety conjugated to the J chain. The heterologous moieties to be linked to a J chain may include, without limitation, a binding moiety, for example, an antibody or an antigen binding fragment. , for example, a single chain Fv (ScFv) molecule, a stabilizing peptide that can increase the half-life of the IgM or IgM-like antibody, or a chemical fraction such as a polymer or a cytotoxin.
[0106] [0106] In some embodiments, a modified J chain may comprise an antigen-binding domain that may include, without limitation, a polypeptide (including small peptides) capable of specifically binding to a target antigen. In certain aspects, an antigen-binding domain associated with a modified J-chain can be an antibody or antigen-binding fragment thereof, as described elsewhere in this document. In some respects, the antigen binding domain can be a scFv antigen binding domain or a single chain antigen binding domain derived, for example, from a camelid or chondricte antibody. The antigen binding domain can be introduced into the J chain at any location that allows the binding of the antigen binding domain to its binding target without interfering with the function of the J chain or the function of an associated IgM or IgA antibody. Insertion locations include, but are not limited to, at or near the C-terminal, at or near the N-terminal, or at the internal location that, based on the three-dimensional structure of the J chain, is accessible. In certain aspects, the antigen binding domain can be introduced into the mature human J chain of SEQ ID NO: 2 between cysteine residues 92 and 101 of SEQ ID NO: 2. In an additional aspect, the antigen binding domain can be introduced into the human J chain of SEQ ID NO: 2 at or near a glycosylation site. In a further aspect, the antigen binding domain can be introduced into the human J chain of SEQ ID NO: 2 within about 10 amino acid residues of the C-terminal or within about 10 amino acids of the N-terminal.
[0107] [0107] IgM or IgM-like pentameric antibodies with mutations in the J chain that alter serum half-life
[0108] [0108] This disclosure provides an IgM antibody or multimerization fragment thereof, for example, an IgM-like pentameric antibody or a multimerization fragment thereof, with increased serum half-life.
[0109] [0109] In certain respects, the variant J chain or its functional fragment, as provided in this document, may have a single amino acid substitution, insertion or deletion, a combination of two single amino acid substitutions, insertions or deletions (for example, two single amino acid substitutions or a single amino acid substitution and a single single amino acid insert or deletion), a combination of three single amino acid substitutions, insertions or deletions, a combination of four single amino acid substitutions, insertions or deletions in that one, two, three, four or more single amino acid substitutions, insertions or deletions may affect, for example, improving the serum half-life of an IgM antibody or IgM-like antibody comprising the variant J chain or its functional fragment. Therefore, an IgM or IgM-like antibody provided may exhibit an increased serum half-life after administration to an animal subject, for example, a mouse model, relative to an IgM or similar IgM-like reference antibody that is identical , except for one or more substitutions, deletions, or single amino acid insertions in the variant J chain or functional fragment thereof, where the supplied antibody and the reference antibody are administered in the same manner to the same animal species.
[0110] [0110] In certain respects, the serum half-life of the supplied IgM or IgM-like antibody, comprising the variant J chain, for example, the α (t1 / 2α) half-life, the β (t1 / 2β half-life) ), both t1 / 2α and t1 / 2β, or the general half-life, can be increased by at least 0.1 times, at least 0.5 times, at least 1 times, at least 5 times, at least 10 times at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 70 times, at least 70 times, at least 90 times, at least 90 times, at least 100 times, at least 500 times at least 1000 times or more on the reference antibody.
[0111] [0111] In certain respects, an IgM antibody or IgM-like antibody comprising a variant J chain, as provided in this document, exhibits further modified pharmacokinetic parameters, for example, a peak peak plasma concentration (Cmax), a increased area under the T0 to curva curve (AUC), for example, a modified clearance time, an increased mean residence time (MRT) or any combination of these in relation to the reference antibody. In some respects, the AUC can be increased by at least 0.1 times, at least 0.5 times, at least 1 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 500 times, at least 1000 times or more than an IgM antibody or similar to the reference IgM which is identical, except for one or more substitutions, deletions or insertions of single amino acid in the variant J chain or its functional fragment, in which the supplied antibody and the reference antibody are administered in the same manner to the same species animals.
[0112] [0112] In certain respects, the J chain of the IgM antibody or IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to the Y102 amino acid of the mature wild-type human J chain (SEQ ID NO: two). "An amino acid corresponding to the Y102 amino acid of the mature wild-type human J chain" means the amino acid in the J chain sequence of any species that is homologous to Y102 in the human J chain. The position corresponding to Y102 in SEQ ID NO: 2 is conserved in the J chain amino acid sequences of at least 43 other species. See FIG. 4 of US Patent No. 9,951,134, which is incorporated by reference in this document. As shown in the Examples, certain mutations at the position corresponding to Y102 of SEQ ID NO: 2 can inhibit the binding of certain immunoglobulin receptors, for example, the human or murine Fcαµ receptor, the murine Fcµ receptor and / or polymeric Ig receptor (receptor of human or murine pIg) to an IgM pentamer comprising the mutant J chain. See, for example, FIG. 2. In certain respects, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by any amino acid. In certain respects, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by alanine (A), serine (S) or arginine (R). In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by alanine. In a particular aspect, the J chain or functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 3. SEQ ID NO: 3 is shown below.
[0113] [0113] In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by serine. In a particular aspect, the J chain or functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 4. SEQ ID NO: 4 is shown below.
[0114] [0114] In a particular aspect, the amino acid corresponding to Y102 of SEQ ID NO: 2 can be replaced by arginine. In a particular aspect, the J chain or functional fragment or variant thereof is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 5. SEQ ID NO: 5 is shown below.
[0115] [0115] In certain respects, the J chain of the IgM antibody or IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to the T103 amino acid of the wild-type mature human J chain (SEQ ID NO: two). "An amino acid corresponding to the wild-type mature human J chain T103 amino acid" means the amino acid in the J chain sequence of any species that is homologous to T103 in the human J chain. The position corresponding to T103 in SEQ ID NO: 2 is conserved in the J chain amino acid sequences of at least 37 other species. See Fig. 4 of US Patent No. 9,951,134, which is incorporated by reference in this document. As demonstrated in the Examples, certain mutations at the position corresponding to T103 in SEQ ID NO: 2 can inhibit the binding of the human Fcαµ receptor to an IgM pentamer comprising the mutant J chain.
[0116] [0116] In certain respects, the isolated variant J chain or functional fragment thereof of the IgM antibody or IgM-like antibody as provided herein comprises an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of the human J chain of mature wild type (SEQ ID NO: 2), provided that S51 is not replaced by threonine (T), or that the J chain comprises amino acid substitutions at the amino acid positions corresponding to amino acids N49 and S51 of the mature human J chain of wild type (SEQ ID NO: 2). Again, "an amino acid corresponding to amino acid N49 of SEQ ID NO: 2 or an amino acid corresponding to S51 of SEQ ID NO: 2 of the mature human J-chain of wild type" means the amino acid in the J chain sequence of any species homologous to N49 and / or S51 in the human J chain. The positions corresponding to N49 and S51 in SEQ ID NO: 2 are conserved in the J chain amino acid sequences of at least 43 other species. See Fig. 4 of US Patent No. 9,951,134, which is incorporated by reference in this document. The amino acids corresponding to N49 and S51 of SEQ ID NO: 2, together with the amino acid corresponding to I50 of SEQ ID NO: 2, comprise an N-glycosylation motif attached to the J chain. Therefore, mutations in N49 and / or S51 (with the exception of a single threonine substitution in S51) may prevent glycosylation for this reason. In certain respects, asparagine at the position corresponding to N49 of SEQ ID NO: 2 can be replaced by any amino acid. In certain respects, asparagine in the position corresponding to N49 of SEQ ID NO: 2 can be replaced by alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect, the position corresponding to the N49 of SEQ ID NO: 2 can be replaced by alanine (A). In a particular aspect, the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 7. SEQ ID NO: 7 is shown below.
[0117] [0117] In certain respects, serine in the position corresponding to S51 of SEQ ID NO: 2 can be replaced by any amino acid except threonine. In certain respects, serine at the position corresponding to S51 of SEQ ID NO: 2 can be replaced by alanine (A) or glycine (G). In a particular aspect, the position corresponding to the S51 of SEQ ID NO: 2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 8. SEQ ID NO: 8 is shown below.
[0118] [0118] In certain respects, the variant J chain or functional fragment thereof of the supplied IgM antibody or IgM-like antibody, as provided herein, is a modified J chain, for example, as provided in the Patent
[0119] [0119] In certain respects, an IgM or IgM-like antibody, as provided herein, comprises a variant J chain or functional fragment thereof, as provided herein, comprising the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 24, or any combination thereof.
[0120] [0120] In certain respects, an IgM or IgM-like antibody comprising a variant J chain, as provided herein, further comprises constant regions of the IgM heavy chain comprising one or more single amino acid substitutions, deletions or insertions with respect to a reference region of the reference IgM heavy chain identical to that of the variant IgM heavy chain constant, except for one or more single amino acid substitutions, deletions or insertions, where the variant IgM heavy chain constant regions can similarly affect half serum life of the supplied IgM antibody or IgM-like antibody. Such an IgM antibody or IgM-like antibody may, in certain respects, exhibit an additionally increased serum half-life after administration to an animal in relation to a reference IgM antibody or an IgM-like antibody which are identical, except for a or more single amino acid substitutions, deletions, or insertions in the constant regions of the IgM heavy chain, and are administered in the same way to the same species of animals. According to this aspect, the reference IgM antibody or IgM-like antibody may be an IgM antibody or IgM-like antibody comprising a variant J chain as provided herein, for example, a variant J chain comprising the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 24, or any combination thereof. In some ways, the increase in serum half-life can be additive, more than additive or less than additive. Exemplary variant IgM heavy chain constant regions are provided elsewhere in this document and include, without limitation, variant human IgM heavy chain constant regions comprising the amino acid sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 31, SEQ ID NO: 32 or SEQ ID NO: 34.
[0121] [0121] In certain respects, the serum half-life of the IgM or IgM-like antibody comprising a variant J chain as provided herein and the regions contained in the variant IgM heavy chain as provided herein comprise an increase in the serum half-life in in relation to a reference antibody comprising only the variant J chain or only the variant IgM constant regions that are additive to the individual increase the serum half-life, are more than additive or less than additive.
[0122] [0122] Variants of the J Chain Affecting the IgM Serum Half-Life
[0123] [0123] This disclosure provides an isolated variant J chain or functional fragment thereof which, as part of a pentameric IgM antibody or a pentameric IgM-like antibody, can increase the serum half-life of that antibody. The supplied variant J chain or functional fragment thereof may be of any kind and comprises one or more substitutions, deletions or insertions of unique amino acids in relation to a reference J chain identical to the variant J chain, except for one or more substitutions, insertions or deletions of unique amino acids. The term "one or more single amino acid substitutions, insertions and deletions" means that each amino acid in the variant J chain amino acid sequence can be individually substituted, deleted or have a single inserted amino acid adjacent to it, but the variant J chain or the functional fragment thereof should still be able to serve the assembly function with IgM heavy chains or IgM-like heavy chains and antibody light chains to form an IgM pentamer or IgM-like pentamer. In certain respects, the variant J chain or its functional fragment, as provided in this document, may have a single amino acid substitution, insertion or deletion, a combination of two single amino acid substitutions, insertions or deletions (for example, two amino acid substitutions single or a single amino acid substitution and a single single amino acid insertion or deletion), a combination of three single amino acid substitutions, insertions or deletions, a combination of four single amino acid substitutions, insertions or deletions or more, where one, two, three, four or more single amino acid substitutions, insertions or deletions can individually or collectively affect the serum half-life of an IgM antibody or IgM-like antibody comprising the variant J chain or its functional fragment.
[0124] [0124] In certain respects, the serum half-life, for example, the half-life α, the half-life β or the general half-life of an IgM or IgM-like antibody comprising the supplied variant J chain or its fragment functional as provided this document can be increased by at least 0.1 times, at least
[0125] [0125] In certain respects, an IgM antibody or IgM-like antibody comprising a variant J chain provided exhibits further modified pharmacokinetic parameters, for example, a peak peak plasma concentration (Cmax), an increased area under the curve from T0 to ∞ (AUC), a modified clearance time, an increased average residence time (MRT) or any combination of these in relation to the reference antibody. In some respects, the AUC can be increased by at least 0.1 times, at least 0.5 times, at least 1 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 500 times, at least 1000 times or more than an IgM antibody or similar to the reference IgM which is identical, except for one or more substitutions, deletions or insertions of single amino acid in the variant J chain or its functional fragment, in which the supplied antibody and the reference antibody are administered in the same manner to the same species animals.
[0126] [0126] In certain respects, the isolated variant J chain or its functional fragment, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to the mature wild type human J chain amino acid Y102 (SEQ ID NO: 2) . "An amino acid corresponding to amino acid Y102 of the mature wild-type human J chain"
[0127] [0127] In certain respects, the isolated variant J chain or its functional fragment, as provided in this document, comprises an amino acid substitution at the amino acid position corresponding to the T103 amino acid of the mature wild-type human J chain (SEQ ID NO: 2) . "An amino acid corresponding to the T103 amino acid of the mature wild-type human J chain" means the amino acid in the J chain sequence of any species that is homologous to T103 in the human J chain. The position corresponding to T103 in SEQ ID NO: 2 is conserved in the J chain amino acid sequences of at least 37 other species. See Fig. 4 of US Patent No. 9,951,134, which is incorporated by reference in this document. As described in the Examples below, amino acid substitutions at the position corresponding to T103 of SEQ ID NO: 2 inhibit the variant J chain from binding to the immunoglobulin Fcαµ receptor. In some respects, the T103 amino acid corresponding to SEQ ID NO: 2 can be replaced by any amino acid. In certain respects, the T103 amino acid corresponding to SEQ ID NO: 2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 6.
[0128] [0128] In certain respects, this disclosure provides an isolated variant J chain or its functional fragment comprises an amino acid substitution at the amino acid position corresponding to the N49 amino acid or the S51 amino acid of the mature human J chain (SEQ ID NO: 2), provided that S51 is not replaced by threonine (T) or where the J chain comprises amino acid substitutions at the amino acid positions corresponding to amino acids N49 and S51 of the mature wild-type human J chain (SEQ ID NO: 2). Again, "an amino acid corresponding to amino acid N49 of SEQ ID NO: 2 of an amino acid corresponding to S51 of SEQ ID NO: 2 of the human wild-type J chain" means the amino acid in the J chain sequence of any species homologous to N49 and / or S51 in the human J chain. The positions corresponding to N49 and S51 in SEQ ID NO: 2 are conserved in the J chain amino acid sequences of at least 43 other species. See Fig. 4 of US Patent No. 9,951,134, which is incorporated by reference in this document. The amino acids corresponding to N49 and S51 of SEQ ID NO: 2, together with the amino acid corresponding to I50 of SEQ ID NO: 2, comprise an N-linked glycosylation motif in the J chain, and mutations in N49 and / or S51 (with exception of a threonine substitution in S51) can thus prevent possible glycosylation in this reason. In certain respects, asparagine at the position corresponding to N49 of SEQ ID NO: 2 can be replaced by any amino acid. In certain respects, asparagine in the position corresponding to N49 of SEQ ID NO: 2 can be replaced by alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect, the position corresponding to the N49 of SEQ ID NO: 2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 7. In certain respects, serine in the position corresponding to S51 of SEQ ID NO: 2 can be replaced by any amino acid (except threonine). In certain respects, serine at the position corresponding to S51 of SEQ ID NO: 2 can be replaced by alanine (A) or glycine (G). In a particular aspect, the position corresponding to the S51 of SEQ ID NO: 2 can be replaced by alanine (A). In a particular aspect, the variant J chain or its functional fragment is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 8.
[0129] [0129] In certain respects, the variant J chain or its functional fragment, as provided in this document, comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 24 or any combination thereof.
[0130] [0130] IgM Constant Regions Variants that confer an increased serum half-life
[0131] [0131] This disclosure further provides an IgM antibody or an IgM-like antibody with increased serum half-life, wherein the IgM antibody or the IgM-like antibody comprises five or six divalent antibody binding units or their variants or fragments, wherein each binding unit comprises two variants of IgM heavy chain constant regions or their multimerizing fragments, each associated with an antigen binding domain or its subunit.
[0132] [0132] In certain respects, a constant region of the variant IgM heavy chain or a multimerization fragment thereof that gives an increased serum half-life to an IgM antibody or IgM-like antibody, as provided in this document, may increase further by half - serum life of a supplied pentameric IgM antibody or an IgM-like antibody with a variant J chain that also increases the serum half-life of the IgM antibody or IgM-like antibody, such as the variant J chains provided elsewhere in this document . In some ways, the increase in serum half-life is additive. In some ways, the increase in serum half-life is more than additive or less than additive.
[0133] [0133] As provided herein, a variant IgM constant region or a multimerization fragment thereof may include one or more single amino acid substitutions, deletions or insertions that affect the serum half-life of an IgM antibody or an IgM-like antibody comprising the variant IgM constant region or fragment. The term "one or more single amino acid substitutions, insertions and deletions" means that each amino acid in the variant IgM constant region or its multimerization fragment can be individually substituted, deleted or have a single inserted amino acid adjacent to it, but the region variant IgM constant or the multimerization fragment thereof must still be able to serve the function within an IgM or IgM-like molecule, for example, antibody, to form an IgM pentamer or hexamer or pentamer or hexamer similar to IgM. In certain respects, the variant IgM constant region or multimerization fragment thereof, as provided in this document, may have a single amino acid substitution, insertion or exclusion, a combination of two single amino acid substitutions, insertions and / or deletions (for example, example, two single amino acid substitutions or a single amino acid substitution and a single amino acid insertion or deletion), a combination of three single amino acid substitutions, insertions and / or deletions, a combination of four substitutions, insertions and / or deletions of single amino acid or more, where one, two, three, four or more isolated single amino acid substitutions, insertions and / or deletions can affect the serum half-life of an IgM antibody or an IgM-like antibody comprising the constant region variant IgM or the multimerization fragment thereof. Therefore, the supplied IgM antibody or IgM-like antibody exhibits an increased serum half-life after administration to an animal in relation to an IgM antibody or a reference IgM-like antibody that are identical, except for one or more single amino acid substitutions, deletions, and / or insertions in the variant IgM constant region or in its multimerization fragment, in which the supplied antibody and the reference antibody are administered in the same manner to the same animal species.
[0134] [0134] In certain respects, the serum half-life of the IgM antibody or the IgM-like antibody, for example, the α half-life, the β half-life or the general half-life, can be increased by at least 0 , 1 times, at least 0.5 times, at least 1 time, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 500 times, at least 1000 or more than the reference antibody. In some ways, the increase in serum half-life is close to that of an IgG antibody that comprises the same antigen-binding domains. In certain respects, a variant IgM constant region or a multimerization fragment thereof can be combined with other antibody modifications
[0135] [0135] In certain respects, an IgM antibody or IgM-like antibody comprising a variant IgM constant region or a multimerization fragment thereof, as provided in this document, exhibits further modified pharmacokinetic parameters, for example, a peak plasma concentration increased peak (Cmax), an increased area under the T0 to curva curve (AUC), a modified clearance time, an increased average residence time (MRT) or any combination of these in relation to the reference antibody.
[0136] [0136] In certain respects, a variant IgM constant region or a multimerization fragment thereof of an IgM antibody or an IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to the S401 amino acid of human IgM constant region of wild type (SEQ ID NO: 12), which in turn corresponds to the amino acid S524 according to the Kabat numbering system. "An amino acid corresponding to the S401 amino acid of the human wild-type IgM constant region" means the amino acid following the IgM constant region of any species that is homologous to S401 in the human IgM constant region. The position corresponding to S401 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species, for example, non-human primates, but not in other species, for example, mouse. See FIG. 1. In certain respects, the S401 of SEQ ID NO: 12 can be replaced by any amino acid. In some ways, SEQ's S401
[0137] [0137] In certain respects, a variant IgM constant region or a multimerization fragment thereof of an IgM antibody or an IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to amino acid E402 of human IgM constant region of wild type (SEQ ID NO: 12), which in turn corresponds to the amino acid E525 according to the Kabat numbering system. "An amino acid corresponding to amino acid E402 of the human wild-type IgM constant region" means the amino acid in the sequence of the IgM constant region of any species that is homologous to E402 in the human IgM constant region. The position corresponding to E402 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species, for example, non-human primates and mice. See FIG. 1. In certain respects, E402 from SEQ ID NO: 12 can be replaced by any amino acid.
[0138] [0138] In certain respects, a variant IgM constant region or a multimerization fragment thereof of an IgM antibody or an IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to amino acid E403 of human IgM constant region of wild type (SEQ ID NO: 12), which in turn corresponds to the amino acid E526 according to the Kabat numbering system. "An amino acid corresponding to amino acid E403 of the human IgM constant region of wild type" means the amino acid in the sequence of the IgM constant region of any species that is homologous to E403 in the human IgM constant region. The position corresponding to E403 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species, for example, non-human primates and mice. See FIG. 1. In certain respects, E403 from SEQ ID NO: 12 can be replaced by any amino acid.
[0139] [0139] In certain respects, a variant IgM constant region or a multimerization fragment thereof of an IgM antibody or an IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to the amino acid R344 of human IgM constant region of wild type (SEQ ID NO: 12), which in turn corresponds to the amino acid R467 according to the Kabat numbering system. "An amino acid corresponding to the R344 amino acid of the human wild-type IgM constant region" means the amino acid in the sequence of the IgM constant region of any species that is homologous to R344 in the human IgM constant region. The position corresponding to R344 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species, for example, non-human primates and mice. See FIG. 1. In certain respects, R344 of SEQ ID NO: 12 can be replaced by any amino acid.
[0140] [0140] In certain respects, a variant IgM constant region or a multimerization fragment thereof of an IgM antibody or an IgM-like antibody, as provided herein, comprises an amino acid substitution at the amino acid position corresponding to amino acid E345 of human IgM constant region of wild type (SEQ ID NO: 12), which in turn corresponds to the amino acid E468 according to the Kabat numbering system. "An amino acid corresponding to amino acid E345 of the human wild-type IgM constant region" means the amino acid in the sequence of the IgM constant region of any species that is homologous to E345 in the human IgM constant region. The position corresponding to E345 in SEQ ID NO: 12 is conserved in the IgM constant region amino acid sequences of some species, for example, non-human primates and mice. See FIG. 1. In certain respects, E345 from SEQ ID NO: 12 can be replaced by any amino acid.
[0141] [0141] Variant Human IgM Constant Regions with Reduced CDC Activity
[0142] [0142] In certain respects, a variant human IgM constant region, when expressed as part of an IgM antibody or an IgM-like antibody, as provided herein, may additionally exhibit reduced complement-dependent cytotoxicity (CDC) activity for cells in the presence of complement to an IgM antibody or an IgM-like reference antibody with an identical corresponding human reference IgM constant region, except for mutations that confer reduced CDC activity. These CDC mutations can be combined with any of the mutations that confer an increased serum half-life, as provided in this document. By "corresponding reference human IgM constant region" is meant a human IgM constant region or a portion thereof, for example, a Cµ3 domain, which is identical to the variant IgM constant region, except for modification or modifications in the constant region that affects CDC activity. In certain aspects, the variant human IgM constant region includes one or more amino acid substitutions, for example, in the Cµ3 domain, with respect to a wild-type human IgM constant region, as described, for example, in the PCT Patent Application PCT / US2018 / 026474, which is incorporated by reference in this document in its entirety. Assays for measuring CDC are well known to those skilled in the art, and exemplary assays are described, for example, in PCT Patent Application No. PCT / US2018 / 026474.
[0143] [0143] In certain respects, a variant human IgM constant region that confers reduced CDC activity includes an amino acid substitution corresponding to the wild type human IgM constant region at position P311 of SEQ ID NO: 12. In other respects, the Variant IgM constant region, as provided in this document, contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO:
[0144] [0144] In one aspect, an IgM antibody or an IgM-like antibody, as provided herein, which includes a variant human IgM constant region comprising amino acid substitutions in P311 and / or P313, for example, P311A, P311S, P311G, P313A, P313S and / or P313G or any combination of these, have a maximum CDC achieved in a dose-response assay reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80% or 90% relative to a binding molecule that includes a corresponding wild-type IgM constant region.
[0145] [0145] The disclosure further provides a polynucleotide, for example, an isolated, recombinant and / or non-naturally occurring polynucleotide, comprising a nucleic acid sequence encoding an IgM or similar IgM antibody polypeptide subunit, as provided herein. By "polypeptide subunit" is meant a portion of a binding molecule, binding unit, IgM antibody, IgM-like antibody or antigen-binding domain that can be translated independently. Examples include, without limitation, an antibody variable domain, for example, a VH or a VL, a J chain, a secretory component, a single chain Fv, an antibody heavy chain, an antibody light chain, a region antibody heavy chain constant, an antibody light chain constant region and / or any fragment, variant or derivative thereof.
[0146] [0146] In certain respects, the polypeptide subunit may comprise an IgM heavy chain constant region or an IgM-like heavy chain constant region or a multimerization fragment thereof, which may be fused to an antigen-binding domain or to a subunit of this, for example, to the VH portion of an antigen-binding domain, all as provided herein. In certain aspects, the polynucleotide can encode a polypeptide subunit comprising a human IgM heavy chain constant region, a human IgM heavy chain constant region, or a multimerization fragment thereof, for example, SEQ ID NO: 12 SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 34, any of which can be fused to an antigen binding domain or a subunit thereof, for example, the C-terminal end of a VH.
[0147] [0147] In certain respects, the polypeptide subunit may comprise an antibody VL portion of an antigen binding domain, as described elsewhere in this document. In certain aspects, the polypeptide subunit can comprise an antibody light chain constant region, for example, a human antibody light chain constant region or a fragment thereof, which can be fused to the C-terminal end of a VL.
[0148] [0148] In certain respects, the polypeptide subunit may comprise a J chain, a modified J chain, or any fragment or functional variant thereof, as provided herein. In certain aspects, the polypeptide subunit can comprise a human J chain or a fragment or functional variant thereof, including modified J chains. In certain respects, the J chain may comprise the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 or SEQ ID NO: 24.
[0149] [0149] In certain respects, a polynucleotide as provided herein, for example, an expression vector such as a plasmid, can include a nucleic acid sequence that encodes a polypeptide subunit, for example, an IgM heavy chain or a heavy chain similar to IgM, a light chain or a J chain, or can include two or more nucleic acid sequences that encode two or more or all three polypeptide subunits of an IgM antibody or an IgM-like antibody as provided herein. Alternatively, the nucleic acid sequences encoding the three polypeptide subunits can be on separate polynucleotides, for example, separate expression vectors. Disclosure provides these vectors of single or multiple expression. The disclosure also provides one or more host cells that encode the polynucleotide (s) or expression vector (s) provided.
[0150] [0150] Thus, to form the antigen-binding domains, nucleic acid sequences that encode the variable regions of antibodies can be inserted into expression vector models for IgM or IgM-like structures, in particular those that encode regions IgM variant heavy chain constants, as provided herein, such as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 34, and can further be combined a polynucleotide encoding a J chain or fragment or functional variant thereof, as provided in this document, for example, encoding SEQ
[0151] [0151] The disclosure further provides a composition comprising two or more polynucleotides, wherein the two or more polynucleotides can collectively encode an IgM or IgM-like antibody with increased serum half-life as described above. In certain aspects, the composition may include a polynucleotide encoding an IgM or wild-type IgM heavy chain or variant, or a multimerization fragment thereof, for example, a human wild-type or variant IgM heavy chain comprising a constant region amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 34, as described above, where the IgM or IgM-like heavy chain further comprises an antigen-binding domain or a subunit thereof, for example, a VH domain. The composition may further include a polynucleotide encoding a light chain or a fragment thereof, for example, a human kappa or lambda light chain comprising at least one VL of an antigen binding domain. A polynucleotide composition as provided may further include a polynucleotide that encodes a J chain, for example, a variant J chain or a functional fragment thereof with at least one single amino acid substitution, insertion or deletion that can enhance the serum half-life of the antibody IgM or similar to IgM, for example, a variant and / or modified J chain comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23 or SEQ ID NO: 24. In certain respects, the polynucleotides that are part of a composition as provided in this document can be located in two, three or more separate vectors, for example, expression vectors. Such vectors are provided by the disclosure. In certain respects, two or more of the polynucleotides that are part of a composition as provided in this document may be located in a single vector, for example, an expression vector. This vector is provided by the disclosure.
[0152] [0152] The disclosure further provides a host cell, for example, a prokaryotic or eukaryotic host cell, comprising a polynucleotide or two or more polynucleotides that encode an IgM or IgM-like antibody as provided herein or any subunit thereof, a polynucleotide composition as provided herein or a vector or two, three or more vectors which collectively encode the IgM or IgM-like antibody as provided herein or any subunit thereof.
[0153] [0153] In a related aspect, the disclosure provides a method of producing an IgM or IgM-like antibody with increased serum half-life, as provided by this disclosure, wherein the method comprises culturing a host cell as provided in this document and the recovery of IgM or IgM-like antibody.
[0154] [0154] J Chain Identification Methods Variants and / or constant regions of IgM variant heavy chain that may confer increased serum half-life
[0155] [0155] This disclosure further provides several methods for identifying subunits of variant antibodies that can increase the serum half-life of an antibody, for example, an IgM antibody or an IgM-like antibody, comprising the subunit. These methods use standard methods of molecular biology and site-directed mutagenesis that will be familiar to those skilled in the art. Exemplary methodologies are provided in the Examples section.
[0156] [0156] In certain respects, this disclosure provides a method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies, pentameric IgM-like antibodies, dimeric IgA antibodies and / or dimeric IgA-like antibodies comprising the chains J variants.
[0157] [0157] In certain respects, this disclosure provides a method for identifying variant J chains that may increase the serum half-life of pentameric IgM antibodies, pentameric IgM-like antibodies, dimeric IgA antibodies and / or dimeric IgA-like antibodies comprising the chains J variants. The method includes testing pentameric IgM antibodies or pentameric IgM-like antibodies or, alternatively, dimeric IgA antibodies or dimeric IgA-like antibodies assembled to include variant J chains or fragments thereof for reduced binding to certain immunoglobulin receptors, for example, the Fc receptor alpha-mu (FcαμR), polymeric Ig receptor (pIgR), Fc-mu receptor (FcµR), two or more receptors, or all three receptors. Regions of the J chain to be subjected to mutagenesis can be targeted by determining those regions required or that contribute to receptor binding, for example, as described in Example 1. Alternatively, amino acids along the entire length of the J chain can be subjected to mutagenesis, and the resulting antibodies comprising the variant J chains can be tested for binding to the receptor. Reduced receptor binding can be measured by any suitable assay, for example, using the ELISA assays described in Example 2.
[0158] [0158] The disclosure further provides a method for identifying IgM variant heavy chain constant regions (or variant IgA heavy chain constant regions) that can increase the serum half-life of an IgM antibody or an IgM-like antibody ( or IgA antibody or IgA-like antibody) comprising the IgM (or IgA) variant heavy chain constant regions. The method for IgM includes testing IgM antibodies or IgM-like antibodies comprising IgM heavy chain constant regions for increased serum half-life in an animal subject against a reference IgM antibody or IgM-like antibody, where the regions Variant IgM heavy chain constants comprise defined amino acid insertions, deletions or substitutions and where the reference IgM antibody or IgM-like antibody comprises IgM heavy chain constant regions identical to the variant IgM heavy chain constant regions, except for defined amino acid insertions, deletions or substitutions.
[0159] [0159] The disclosure further provides a method for identifying IgM variant heavy chain constant regions (or, alternatively, variant IgA heavy chain constant regions) that can increase the serum half-life of an IgM antibody or a similar antibody IgM (or an IgA antibody or an IgA-like antibody) comprising the variant heavy chain constant regions. The method for IgM includes testing IgM antibodies or IgM-like antibodies comprising IgM heavy chain constant regions for reduced binding to the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcµR), polymeric Ig receptor (pIgR), any combination of two of the receptors or all three receptors with respect to a reference IgM antibody or IgM-like antibody, where the variant IgM heavy chain constant regions comprise defined amino acid insertions, deletions or substitutions and where the IgM antibody The reference or IgM-like antibody comprises IgM heavy chain constant regions identical to variant IgM heavy chain constant regions, except for defined amino acid insertions, deletions or substitutions. The method further includes recovering IgM antibodies or IgM-like antibodies comprising variant IgM heavy chain constant regions that confer reduced FcαµR binding capacity, reduced FcµR binding capacity, reduced pIgR binding capacity, binding capacity to any two receptors reduced or ability to bind to all three receptors reduced in IgM antibodies or in IgM-like antibodies comprising variant IgM heavy chain constant regions relative to the reference IgM or IgM-like antibody. Any number of IgM heavy chain constant or IgM-like variant regions comprising, for example, one, two, three, four or more substitutions,
[0160] [0160] The disclosure further provides a method of treating a disease or disorder in a subject in need of treatment comprising administering to the subject a therapeutically effective amount of an IgM or IgM-like antibody as provided herein. By "therapeutically effective dose or amount" or "effective amount" is meant an amount of an IgM or IgM-like antibody, which when administered causes a positive immunotherapeutic response in relation to the subject's treatment.
[0161] [0161] Effective doses of cancer treatment compositions vary depending on several factors, including the means of administration, target site, subject's physiological state, whether the subject is human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic.
[0162] [0162] The subject to be treated can be any animal, for example, mammal, in need of treatment; in some ways, the subject is a human subject.
[0163] [0163] In its simplest form, a preparation to be administered to a subject is an IgM or IgM-like antibody as provided herein, or a fragment of binding to the multimeric antigen of that administered in the conventional dosage form, which can be combined with a pharmaceutical excipient, carrier or diluent as described elsewhere in this document.
[0164] [0164] The compositions of the disclosure can be administered by any suitable method, for example, parenterally, intraventricular or orally, by inhalation spray, topically, rectally, nasal, buccal or vaginally, or through an implanted reservoir . The term "parenteral", as used in this document, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, cholestasis, intralesional and intracranial injection or infusion techniques.
[0165] [0165] The methods of preparing and administering an IgM or IgM-like antibody as provided herein to a subject in need of it are well known or are readily determined by those skilled in the art in view of this disclosure. The route of administration can be, for example, intratumor, oral, parenteral, inhalation or topical. The term "parenteral", as used herein, includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although these forms of administration are contemplated as suitable forms, another example of a form of administration would be an injectable solution, in particular for intratumor, intravenous or intra-arterial injection or drip. An appropriate pharmaceutical composition can comprise a buffer (for example, acetate, phosphate or citrate buffer), a surfactant (for example, polysorbate), optionally a stabilizing agent (for example, human albumin), etc.
[0166] [0166] As provided herein, an IgM or IgM-like antibody as provided herein may be administered in a pharmaceutically effective amount for the treatment of a subject in need. In this regard, it will be appreciated that the IgM or IgM-like antibodies disclosed can be formulated so as to facilitate administration and promote the stability of the active agent. The pharmaceutical compositions can comprise a sterile, non-toxic, pharmaceutically acceptable carrier such as physiological saline, non-toxic buffers, preservatives and the like. A pharmaceutically effective amount of an IgM or IgM-like antibody as provided herein means an amount sufficient to obtain effective binding to a target and obtain a therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences, for example, 21st Edition (Lippincott Williams & Wilkins) (2005).
[0167] [0167] Certain pharmaceutical compositions provided in this document can be administered orally in an acceptable dosage form including, for example, capsules, tablets, suspensions or aqueous solutions. Certain pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline solution, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability and / or other conventional agents of solubilization or dispersion.
[0168] [0168] The amount of an IgM or IgM-like antibody that can be combined with carrier materials to produce a single dosage form will vary depending, for example, on the treated subject and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens can be adjusted to provide the ideal desired response (for example, a therapeutic or prophylactic response).
[0169] [0169] According to the scope of the present disclosure, an IgM or IgM-like antibody as provided herein can be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect. An IgM or IgM-like antibody as provided herein may be administered to the subject in a conventional dosage form prepared by combining the antibody or a fragment of antigen-binding, variant or multimeric derivative thereof, according to the disclosure, with a carrier or conventional pharmaceutically acceptable diluents according to known techniques. The form and characteristics of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which they are to be combined, the route of administration and other well-known variables.
[0170] [0170] This disclosure also provides for the use of an IgM or IgM-like antibody as provided herein in the manufacture of a drug for the treatment, prevention or control of cancer.
[0171] [0171] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the capabilities of the technique. These techniques are fully explained in the literature. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed .; Cold Spring Harbor Laboratory Press); Sambrook et al., Ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2nd Edition (IRL Press), Volumes 1-4; Gait, ed.
[0172] [0172] The general principles of antibody manipulation are presented,
[0173] [0173] All references cited above, as well as all references cited in this document, are incorporated in this document by reference in their entirety.
[0174] [0174] The following examples are offered by way of illustration and not by way of limitation. Examples
[0175] [0175] Our hypothesis was that the interaction between IgM and one or more of its Ig receptors, the Fc µ receptor (FcμR), the Fc α / µ receptor (FcαμR) and / or the polymeric Ig receptor (pIgR) contributed to the specific pharmacokinetics and pharmacodynamics observed for IgM antibodies in vivo. Therefore, we proposed to identify specific regions of IgM involved in binding to these receptors and asked whether changes in those regions, for example, amino acid substitutions, could modulate, for example, inhibit binding to the receptor for an IgM antibody comprising the changes. We then tested whether any of these changes affected the in vivo plasma availability of IgM antibodies comprising the changes.
[0176] [0176] Example 1: Screening of J Chain and Fcµ Peptide Arrangements Regarding Receptor Binding
[0177] [0177] To identify the regions in the IgM constant regions and the J chain where FcμR, FcαμR and / or pIgR bind, we use a technology based on peptide matrix (PEPperPRINT; WWW_dot_pepperprint_dot_com). A complete set of peptides based on the human J chain amino acid sequence (SEQ ID NO: 2) and the human IgM Cµ3 and Cµ4 domains (around amino acid 224 to about amino acid 430 of SEQ ID NO: 12) were synthesized and immobilized on solid supports, as follows. IgM µ3, µ4 and J chain sequences were stretched with neutral GSGSGSG ligands (SEQ ID NO: 33) at the N and C terminals to avoid truncated peptides. The elongated protein sequences were translated as 9 and 13 amino acid peptides with peptide-peptide overlays of 8 and 12 amino acids, respectively. After peptide synthesis, all peptides were cyclized through a thioether bond between a thiol group on the C-terminal cysteine side chain and an appropriately modified N-terminal. The resulting IgM peptide microarrays contained 844 different peptides printed in duplicate (1,688 peptide stains), along with control peptides. Restricted peptides with increments of one residue were analyzed with 1, 10 or 100 µg / mL of recombinant human pIgR (amino acids 19-638 from SEQ ID NO: 20, available from R&D Systems), human FcμR (amino acids 18-251 from SEQ ID NO: 21, available from R&D Systems) and human FcαμR (amino acids 17 to 450 from SEQ ID NO: 22, available from R&D Systems), each with a 6X HIS tag in incubation buffer (PBS, pH 7.4 with 0.005% Tween 20 and 10% Rockland blocking buffer (MB-070)) was followed by staining with secondary mouse anti-6x-His Epitope Tag DyLight680 antibody and with the control antibodies, as well as reading at the intensities of 7/7 scan (red green).
[0178] [0178] Stain intensities quantification and peptide annotation were based on 16-bit grayscale tiff files at 7/7 scan intensities that exhibit a higher dynamic range than 24-bit color tiff files ; microarray image analysis was performed with PEPSLIDE® Analyzer. A software algorithm divides the fluorescence intensities of each spot into a raw signal, a foreground signal and a background signal (see the "Raw Data" tabs) and calculates the average foreground intensities and the spot-to-spot deviations of the stain duplicates. Based on the calculated average foreground intensities, an intensity map was generated.
[0179] [0179] The average spot intensities of the assays were plotted with the target proteins against the protein sequence from the N-terminal of the IgM µ3 chain to the C-terminal of the IgM J chain to view the general spot intensities and signal-to-signal ratios. noise. The intensity plots were correlated with peptide and intensity maps, as well as with visual inspection of the microarray scans to identify the main interactions of the target proteins.
[0180] [0180] Using the raw data, key regions of the J chain were identified, as well as HCM IgM constant regions that bound to the receptors on the PEPperPRINT platform.
[0181] [0181] Human FcαμR linked to the following underlined regions of the mature J chain, SEQ ID NO: 2: 1 QEDERIVLVD NKCKCARITS RIIRSSEDPN EDIVERNIRI IVPLNNRENI 51 SDPTSPLRTR FVYHLSDLCK KCDPTEVELD NQIVTATQSN
[0182] [0182] Therefore, regions of the mature human J chain that contribute to the binding of human FcαμR may include, for example, amino acids within and / or amino acids overlapping and / or amino acids comprising,
[0183] [0183] human pIgR linked to the following doubly underlined regions of the mature J chain, SEQ ID NO: 2: 1 QEDERIVLVD NKCKCARITS RIIRSSEDPN EDIVERNIRI IVPLNNRENI 51 SDPTSPLRTR FVYHLSDLCK KCDPTEVELD NQIVTATQSN
[0184] [0184] Accordingly, regions of the mature human J chain that contribute to the binding of human plgR may include, for example, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 12 to 24 of SEQ ID NO: 2, and / or amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 93 to 105 of SEQ ID NO: 2. As those of ordinary skill in the art will appreciate, IgM antibodies or pentameric IgM-like antibodies that comprise variant J chains with mutations, for example, amino acid insertions, deletions and / or substitutions in these regions, superimposed on those regions and / or corresponding to those regions (for example, in an amino acid sequence of J chain of another species), can be tested for inhibition of pIgR binding, for example, as described in Example 2, and / or for serum life, for example, as described in Example 3.
[0185] [0185] Human FcμR linked to the following rippled regions of the mature J chain, SEQ ID NO: 2: 1 QEDERIVLVD NKCKCARITS RIIRSSEDPN EDIVERNIRI IVPLNNRENI 51 SDPTSPLRTR FVYHLSDLCK KCDPTEVELD NQIVTATQSN
[0186] [0186] Therefore, regions of the mature human J chain that contribute to the binding of human FcμR may include, for example, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 1 to 4 of SEQ ID NO: 2, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 87 to 105 of SEQ ID NO: 2, and / or amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 125 to 137 of SEQ ID NO: 2. As those commonly skilled in the art will appreciate, IgM antibodies or pentameric IgM-like antibodies comprising variant J chains with mutations, for example, insertions, deletions and / or substitutions of amino acids in these regions, overlapping these regions and / or corresponding to those regions (for example, in an amino acid sequence those of J chain of another species), can be tested for inhibition of FcμR binding, for example, as described in Example 2, and / or for increased serum half-life, for example, as described in Example 3.
[0187] [0187] Human FcαμR linked to the following underlined Cμ3 and Cμ4 regions of the human IgM constant region, SEQ ID NO: 12 (numbering for SEQ ID NO: 12 / KABAT provided):
[0188] [0188] Sequential numbering key (SEQ ID NO: 12) / KABAT for IgM heavy chain 1/127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF
[0189] [0189] Therefore, regions of the human IgM constant region that contribute to the binding of human FcαμR may include, for example, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 241 to 253 of SEQ ID NO: 12, amino acids within and / or amino acids overlapping and / or amino acids comprising, consisting of or consisting essentially of amino acids 282 to 294 of SEQ ID NO: 12, amino acids within and / or amino acids overlapping and / or amino acids comprising , consisting of or consisting essentially of amino acids 342 to 354 of SEQ ID NO: 12, and / or amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 393 to 415 of SEQ ID NO: 12 As those of ordinary skill in the art will appreciate, IgM antibodies or IgM-like antibodies that comprise IgM heavy chain constant regions with mut actions, for example, insertions, deletions and / or substitutions of amino acids in these regions, overlapping these regions and / or corresponding to those regions (for example, in a sequence of amino acids of constant region of IgM heavy chain of another species, as those shown in FIG. 1), can be tested for inhibition of FcαμR binding, for example, as described in Example 2, and / or for increased serum half-life, for example, as described in Example 3.
[0190] [0190] human pIgR linked to the following double-underlined Cμ3 and Cμ4 regions of the human IgM constant region, SEQ ID NO: 12 (numbering for SEQ ID NO: 12 / KABAT provided): 1/127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITFF
[0191] [0191] Therefore, regions of the human IgM constant region that contribute to the binding of human plgR may include, for example, amino acids within and / or overlapping amino acids and / or amino acids comprising, consisting of or consisting essentially of amino acids 232 to 244 of SEQ ID NO: 12, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 287 to 304 of SEQ ID NO: 12, and / or amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 397 to 413 of SEQ ID NO: 12. As those of ordinary skill in the art will appreciate, IgM antibodies or IgM-like antibodies that comprise mutated IgM heavy chain constant regions, for example , insertions, deletions and / or substitutions of amino acids in these regions, overlapping those regions and / or corresponding to those regions (for example, in an IgM heavy chain constant region amino acid sequence of another species, as shown in FIG. 1), can be tested for inhibition of plgR binding, for example, as described in Example 2, and / or for increased serum half-life, for example, as described in Example 3.
[0192] [0192] Human FcμR linked to the following wavy underlined Cμ3 and Cμ4 regions of the human IgM constant region, SEQ ID NO: 12 (numbering for SEQ ID NO: 12 / KABAT provided): 1/127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF
[0193] [0193] Therefore, regions of the human IgM constant region that contribute to the binding of human FcμR may include, for example, amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 282 to 305 of SEQ ID NO: 12, and / or amino acids within and / or amino acids superimposed on and / or amino acids comprising, consisting of or consisting essentially of amino acids 393 to 417 of SEQ ID NO: 12. As those commonly skilled in the art will appreciate, antibodies IgM or IgM-like antibodies that comprise IgM heavy chain constant regions with mutations, for example, amino acid insertions, deletions and / or substitutions in those regions, overlapping these regions and / or corresponding to those regions (for example, in a IgM heavy chain amino acid sequence of another species, such as those shown in FIG. 1), can be tested for inhibition of FcμR, for example, as described in Example 2, and / or regarding the increase in serum half-life, for example, as described in Example 3.
[0194] [0194] Example 2: Construction of the alanine scan of IgM and Fc J-chain mutants and screening for inhibition of receptor binding by ELISA
[0195] [0195] The J chain and IgM constant regions of the monospecific IgM anti-CD20 antibody 1.5.3 as a monospecific pentamer (with the mature human J chain of SEQ ID NO: 2) and the bispecific IgM anti-CD20 antibody 1.5. 3 IgM as a bispecific pentamer (with the J chain "V15J", SEQ ID NO: 9), both as described in PCT Publ. No. WO 2016/141303, underwent mutagenesis alanine scanning to determine whether certain regions identified by PEPperPRINT analysis (Example 1) could be modified to reduce or inhibit the binding of FcαμR, FcμR and / or pIgR. The tested mutants included substitutions of J-chain alanine at positions corresponding to positions Y102 and T103 of the mature human J-chain of SEQ ID NO: 2 (Y102A and T103A). The modified J "V15J" chain comprising the Y102A mutation is shown as SEQ ID NO: 10, the modified J "V15J" chain comprising the T103A mutation is shown as
[0196] [0196] Mutants tested in the human IgM constant region included alanine mutations at positions corresponding to R344, E345, S401 and E402 of SEQ ID NO: 12 (R344A, SEQ ID NO: 31, E345A, SEQ ID NO: 32, S401A, SEQ ID NO: 13, E402A, SEQ ID NO: 14 and E403A, SEQ ID NO: 34). Two mutations in the variant IgM constant region, SEQ ID NO: 13 and SEQ ID NO: 14, were tested for their effect on the in vivo half-life of IgMs comprising the mutations, with the "wild type" J V15J chain ( SEQ ID NO: 9) and with the J chain V15J comprising the Y102A mutation (SEQ ID NO: 10), see Example 3).
[0197] [0197] The DNA fragments encoding the heavy, light and J chains of the selected mutants were synthesized by a commercial supplier. The DNA constructs were transformed into competent bacteria and plated on LB plates with multiple selective antibiotics. Several bacterial colonies were chosen, and DNA preparations were made using standard molecular biology techniques.
[0198] [0198] Binding to the receptor for some of the IgM mutants and the corresponding IgM and IgG controls was measured by ELISA as follows.
[0199] [0199] The binding of several mutant IgMs to human and mouse receptors is summarized in FIG. 2. The T103A mutation in the J chain inhibited binding to human FcαμR, but not to human pIgR or human FcμR (data not shown).
[0200] [0200] Bispecific anti-CD20 / anti-CD3 antibodies with additional amino acid substitutions on the modified J chain V15J at the amino acid position corresponding to Y102 of SEQ ID NO: 2 were also evaluated for their effect on pIgR receptor binding. Tyrosine at position 102 was replaced by aspartic acid (Y102D), phenylalanine (Y102F), arginine (Y102R), serine (Y102S) and threonine
[0201] [0201] Example 3: Pharmacokinetic analysis of anti-CD20 antibodies comprising selected mutations that affect receptor binding
[0202] [0202] This example demonstrates that certain amino acid substitutions in the J chain or in the IgM constant region of monospecific or bispecific IgM pentameric antibodies can increase the serum half-life of these antibodies. The J chain and IgM constant regions of the monospecific IgM anti-CD20 antibody 1.5.3 as a monospecific pentamer (with the mature J chain of SEQ ID NO: 2) and the bispecific IgM anti-CD20 antibody 1.5.3 as a pentamer bispecific (with J chain “V15J”, SEQ ID NO: 9), both as described in PCT Publ. No. WO 2016/141303, were submitted to alanine scan mutagenesis and tested for changes in pharmacokinetic parameters in mice. The tested mutants included substitutions of J-chain alanine at positions corresponding to positions Y102 and T103 of the mature human J-chain of SEQ ID NO: 2 (Y102A and T103A). The modified "V15J" J chain comprising the Y102A mutation is shown as SEQ ID NO: 10.
[0203] [0203] Pharmacokinetic parameters were measured for various IgM antibodies in a mouse model in vivo as follows. Balb / c mice were injected with 100 µg of each antibody by intravenous infusion.
[0204] [0204] FIG. 5 shows the serum concentrations over time of some of the bispecific mutant IgM antibodies compared to an IgG antibody comprising the same VH and VL regions. As shown by these results, the serum half-life of IgMs with a single Y102A mutation on the J chain or with the Y102A mutation plus an additional mutation on the IgM heavy chain approximates that of a comparable IgG antibody.
[0205] [0205] Example 4: Extension of the half-life of the IgM antibody molecule comprising the human J chain of variant N49A.
[0206] [0206] This example demonstrates that another single amino acid substitution in the human J chain can increase the serum half-life of an IgM antibody comprising the variant J chain. A modified J chain described above comprising a mature human J chain with a heterologous antigen-binding domain that binds to CD3 fused to its N-terminus through a 15 amino acid linker ("V15J", SEQ ID NO: 9, see US Patent 9,951,134) was mutated using standard techniques to introduce an alanine substitution at the position corresponding to N49 of the mature human wild-type J chain (SEQ
[0207] [0207] This J chain construct was used to produce 1.5.3 V15J and 1.5.3 V15J-N49A, exemplary pentameric IgM antibodies that bind to CD20. The CD20 antigen binding domain and methods for producing the IgM antibody have been described in PCT Publ. WO 2016/141303. The antibodies were tested for proper assembly, antigen binding and the ability of CD3 ligands to activate T cells.
[0208] [0208] Pharmacokinetic studies (PK) were performed on Balb / c mice, as described in Example 3, to assess clearance of IgM antibodies.
[0209] [0209] The results are shown in FIG. 6. As shown, IgM
[0210] [0210] The scope and scope of this disclosure should not be limited by any of the examples of modalities described above, but should be defined only in accordance with the following claims and their equivalents.

权利要求:
Claims (79)
[1]
1. IgM antibody or an IgM-like antibody with increased serum half-life CHARACTERIZED by the fact that it comprises five units of binding to bivalent antibodies or variants or fragments thereof and a variant J chain or functional fragment thereof, where each unit of The linkage comprises two IgM heavy chain constant regions or multimerization fragments or variants thereof, each associated with an antigen binding domain or a subunit thereof, where the variant J chain or a functional fragment thereof comprises one or more substitutions , single amino acid deletions or insertions relative to a reference J chain identical to the variant J chain, except for one or more single amino acid substitutions or insertions, and where the variant J chain can affect the serum half-life of the IgM antibody or IgM-like antibody; and wherein the supplied IgM antibody or the IgM-like antibody exhibits an increased serum half-life after administration to an animal subject to a reference IgM antibody or an IgM-like antibody that are identical except for one or more substitutions, deletions, or insertions of unique amino acids in the variant J chain and are administered in the same way to the same animal species.
[2]
2. IgM antibody or IgM-like antibody according to claim 1, CHARACTERIZED by the fact that the variant J chain or a functional fragment thereof comprises one, two, three or four substitutions, deletions or insertions of single amino acid in relation to reference J chain.
[3]
3. IgM antibody or IgM-like antibody according to claim 1 or claim 2, CHARACTERIZED by the fact that the variant J chain or a functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the chain
Human wild-type J (SEQ ID NO: 2).
[4]
4. IgM antibody or IgM-like antibody according to claim 3, CHARACTERIZED by the fact that the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by alanine (A), serine (S) or arginine (R) .
[5]
5. IgM antibody or IgM-like antibody according to claim 4, CHARACTERIZED by the fact that the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by alanine (A).
[6]
6. IgM antibody or IgM-like antibody according to claim 5, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 3.
[7]
7. IgM antibody or IgM-like antibody according to claim 4, CHARACTERIZED by the fact that the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by serine (S).
[8]
8. IgM antibody or IgM-like antibody according to claim 7, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 4.
[9]
9. IgM antibody or IgM-like antibody according to claim 4, CHARACTERIZED by the fact that the amino acid corresponding to Y102 of SEQ ID NO: 2 is replaced by arginine (R).
[10]
10. IgM antibody or IgM-like antibody according to claim 9, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 5.
[11]
11. IgM antibody or IgM-like antibody according to claim 1 or claim 2, CHARACTERIZED by the fact that the variant J chain or a functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to the T103 amino acid of the J chain human type (SEQ ID NO: 2).
[12]
12. IgM antibody or IgM-like antibody according to claim 11, CHARACTERIZED by the fact that the amino acid corresponding to T103 of SEQ ID NO: 2 is replaced by alanine (A).
[13]
13. IgM antibody or IgM-like antibody according to claim 12, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 6.
[14]
14. IgM antibody or IgM-like antibody according to claim 1 or claim 2, CHARACTERIZED by the fact that the variant J chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of the human J chain (SEQ ID NO: 2), where S51 is not substituted by threonine (T) or where the variant J chain comprises amino acid substitutions at the amino acid positions corresponding to amino acids N49 and S51 of the human J chain ( SEQ ID NO: 2).
[15]
15. IgM antibody or IgM-like antibody according to claim 14, CHARACTERIZED by the fact that the position corresponding to N49 of SEQ ID NO: 2 is replaced by alanine (A), glycine (G), threonine (T) , serine (S) or aspartic acid (D).
[16]
16. IgM antibody or IgM-like antibody according to claim 15, CHARACTERIZED by the fact that the position corresponding to N49 of SEQ ID NO: 2 is replaced by alanine (A).
[17]
17. IgM antibody or IgM-like antibody according to claim 16, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 7.
[18]
18. IgM antibody or IgM-like antibody according to claim 14, CHARACTERIZED by the fact that the position corresponding to S51 of SEQ ID NO: 2 is replaced by alanine (A) or glycine (G).
[19]
19. IgM antibody or IgM-like antibody according to claim 18, CHARACTERIZED by the fact that the position corresponding to S51 of SEQ ID NO: 2 is replaced by alanine (A).
[20]
20. IgM antibody or IgM-like antibody according to claim 19, CHARACTERIZED by the fact that the J chain is a variant human J chain and comprises the amino acid sequence SEQ ID NO: 8.
[21]
21. IgM antibody or IgM-like antibody according to any one of claims 1 to 20, CHARACTERIZED by the fact that the IgM heavy chain constant regions or their multimerization fragments are variant IgM heavy chain constant regions that comprise one or more single amino acid substitutions, deletions or insertions with respect to a reference IgM heavy chain constant region identical to the variant IgM heavy chain constant regions, except for one or more single amino acid substitutions, deletions or insertions, where variant IgM heavy chain constant regions can affect the serum half-life of the IgM antibody or IgM-like antibody, and where the IgM antibody or IgM-like antibody exhibits an additionally increased serum half-life after administration to an animal subject to an IgM antibody or reference IgM-like antibody that is identical, except for one or more substitutions, del single amino acid reactions or insertions in the IgM heavy chain constant regions and is administered in the same way to the same animal species.
[22]
22. IgM antibody or IgM-like antibody according to claim 21, CHARACTERIZED by the fact that the additional increase in serum half-life is additive.
[23]
23. IgM antibody or IgM-like antibody with increased serum half-life CHARACTERIZED by the fact that it comprises five or six divalent antibody binding units or variants or fragments thereof,
wherein each binding unit comprises two IgM variant heavy chain constant regions or multimerization fragments thereof, each associated with an antigen binding domain or a subunit thereof, wherein the variant IgM heavy chain constant regions or their multimerization fragments comprise one or more single amino acid substitutions, deletions or insertions with respect to a reference IgM heavy chain constant region identical to the variant IgM heavy chain constant regions, except for one or more substitutions, deletions or insertions of single amino acid, and where the constant regions of the variant IgM heavy chain can affect the serum half-life of the IgM antibody or IgM-like antibody; and wherein the supplied IgM antibody or IgM-like antibody exhibits an increased serum half-life after administration to an animal subject to a reference IgM antibody or IgM-like antibody that are identical, except for one or more substitutions , deletions, or insertions of unique amino acids in the constant regions of the IgM heavy chain, and are administered in the same way to the same species of animals.
[24]
24. IgM antibody or IgM-like antibody according to any of claims 21 to 23, CHARACTERIZED by the fact that variant IgM heavy chain constant regions comprise one, two, three or four substitutions, deletions or insertions of unique amino acid in relation to the reference IgM heavy chain constant region.
[25]
25. IgM antibody or IgM-like antibody according to any one of claims 21 to 24, CHARACTERIZED by the fact that the IgM variant heavy chain constant regions comprise an amino acid substitution at the amino acid position corresponding to the amino acid R344 of human IgM constant region wild type SEQ ID NO: 12
[26]
26. IgM antibody or IgM-like antibody according to claim 25, CHARACTERIZED by the fact that the amino acid corresponding to R344 of SEQ ID NO: 12 is replaced by alanine (A).
[27]
27. IgM antibody or IgM-like antibody according to claim 26, CHARACTERIZED by the fact that variant IgM heavy chain constant regions are variant human IgM heavy chain constant regions and comprise the amino acid sequence SEQ ID NO : 31.
[28]
28. IgM antibody or IgM-like antibody according to any one of claims 21 to 24, CHARACTERIZED by the fact that variant IgM heavy chain constant regions comprise an amino acid substitution at the amino acid position corresponding to amino acid E345 of human IgM constant region of wild type SEQ ID NO: 12.
[29]
29. IgM antibody or IgM-like antibody according to claim 28, CHARACTERIZED by the fact that the amino acid corresponding to E345 of SEQ ID NO: 12 is replaced by alanine (A).
[30]
30. IgM antibody or IgM-like antibody according to claim 29, CHARACTERIZED by the fact that variant IgM heavy chain constant regions are variant human IgM heavy chain constant regions and comprise the amino acid sequence SEQ ID NO : 32.
[31]
31. IgM antibody or IgM-like antibody according to any of claims 21 to 24, CHARACTERIZED by the fact that variant IgM heavy chain constant regions comprise an amino acid substitution at the amino acid position corresponding to amino acid S401 of human IgM constant region of wild type SEQ ID NO: 12.
[32]
32. IgM antibody or IgM-like antibody according to claim 31, CHARACTERIZED by the fact that the amino acid corresponding to S401 of SEQ ID NO: 12 is replaced by alanine (A).
[33]
33. IgM antibody or IgM-like antibody according to claim 32, CHARACTERIZED by the fact that variant IgM heavy chain constant regions are variant human IgM heavy chain constant regions and comprise the amino acid sequence SEQ ID NO : 13.
[34]
34. IgM antibody or IgM-like antibody according to any one of claims 21 to 24, CHARACTERIZED by the fact that variant IgM heavy chain constant regions comprise an amino acid substitution at the amino acid position corresponding to amino acid E402 of human IgM constant region of wild type SEQ ID NO: 12.
[35]
35. IgM antibody or IgM-like antibody according to claim 34, CHARACTERIZED by the fact that the amino acid corresponding to E402 of SEQ ID NO: 12 is replaced by alanine (A).
[36]
36. IgM antibody or IgM-like antibody according to claim 35, CHARACTERIZED by the fact that variant IgM heavy chain constant regions are variant human IgM heavy chain constant regions and comprise the amino acid sequence SEQ ID NO : 14.
[37]
37. IgM antibody or IgM-like antibody according to any one of claims 21 to 24, CHARACTERIZED by the fact that variant IgM heavy chain constant regions comprise an amino acid substitution at the amino acid position corresponding to amino acid E403 of human IgM constant region of wild type SEQ ID NO: 12.
[38]
38. IgM antibody or IgM-like antibody according to claim 37, CHARACTERIZED by the fact that the amino acid corresponding to E403 of SEQ ID NO: 12 is replaced by alanine (A).
[39]
39. IgM antibody or IgM-like antibody according to claim 38, CHARACTERIZED by the fact that variant IgM heavy chain constant regions are variant human IgM heavy chain constant regions and comprise the amino acid sequence SEQ ID NO : 34.
[40]
40. IgM antibody or IgM-like antibody according to any one of claims 1 to 39, CHARACTERIZED by the fact that the increased serum half-life comprises an increased alpha half-life (t1 / 2α), a half-life increased beta (t1 / 2β) or an increased t1 / 2α and an increased t1 / 2β.
[41]
41. IgM antibody or IgM-like antibody according to any one of claims 1 to 40, CHARACTERIZED by the fact that it still exhibits an increased plasma concentration peak (Cmax), an area under the increased curve (AUC), an modified clearance time or any combination of these relative to the reference antibody.
[42]
42. IgM antibody or IgM-like antibody according to any one of claims 1 to 41, CHARACTERIZED by the fact that IgM heavy chain constant regions or multimerization fragments or variants thereof comprise a Cµ4 domain and a tail ( tp) of IgM.
[43]
43. IgM antibody or IgM-like antibody according to claim 42, CHARACTERIZED by the fact that IgM heavy chain constant regions or multimerization fragments or variants thereof further comprise a Cµ3 domain, a Cµ2 domain, a Cµ1 domain or any combination of these.
[44]
44. IgM antibody or IgM-like antibody according to any one of claims 1 to 43, CHARACTERIZED by the fact that the antigen binding domain comprises a single chain Fv fragment (ScFv) or a single domain variable region (VHH).
[45]
45. IgM antibody or IgM-like antibody according to any of claims 1 to 43, CHARACTERIZED by the fact that the subunit of the antigen-binding domain comprises a variable region of heavy chain (VH).
[46]
46. IgM antibody or IgM-like antibody according to any one of claims 1 to 45, CHARACTERIZED by the fact that each binding unit further comprises two constant regions of light chain or fragments or variants thereof, each associated with a antigen-binding domain a subunit of this.
[47]
47. IgM antibody or IgM-like antibody according to claim 46, CHARACTERIZED by the fact that the light chain constant regions are kappa or lambda light chain constant regions or fragments or variants thereof, and in which the binding domain the antigen comprises a ScFv fragment or the subunit of the antigen binding domain comprises a light chain variable region (VL).
[48]
48. IgM antibody or IgM-like antibody according to any one of claims 1 to 47, CHARACTERIZED by the fact that the J chain or a functional fragment or a variant thereof comprises one or more heterologous polypeptides directly or indirectly fused to J chain or a functional fragment or variant thereof.
[49]
49. IgM antibody or IgM-like antibody according to claim 48, CHARACTERIZED by the fact that one or more heterologous polypeptides are fused to the J chain or to a fragment of this pathway a peptide linker.
[50]
50. IgM antibody or IgM-like antibody according to claim 49, CHARACTERIZED by the fact that the peptide linker comprises at least 5 amino acids, but not more than 25 amino acids.
[51]
51. IgM antibody or IgM-like antibody according to claim 50, CHARACTERIZED by the fact that the peptide linker consists of GGGGS (SEQ ID NO: 25), GGGGSGGGGS (SEQ ID NO: 26), GGGGSGGGGSGGGS (SEQ ID NO : 27), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 29).
[52]
52. IgM antibody or IgM-like antibody according to claims 48 to 51, CHARACTERIZED by the fact that the one or more heterologous polypeptides are fused to the N-terminus of the J chain or to a fragment or variant thereof, to the C-terminal of the J chain or to a fragment or variant thereof, or in which the heterologous polypeptides are fused to the N-terminal and to the C-terminal of the J chain or to a fragment or variant thereof, in which the heterologous polypeptides can be the same or different.
[53]
53. IgM antibody or IgM-like antibody according to claims 48 to 52, CHARACTERIZED by the fact that at least one heterologous polypeptide comprises a binding domain.
[54]
54. IgM antibody or IgM-like antibody according to claim 53, CHARACTERIZED by the fact that the heterologous polypeptide binding domain is an antibody or antigen binding fragment thereof.
[55]
55. IgM antibody or IgM-like antibody according to claim 54, CHARACTERIZED by the fact that the antigen-binding fragment comprises a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, an Fd fragment , an Fv fragment, a single chain Fv fragment (scFv), a disulfide-bound Fv fragment (sdFv) or any combination thereof.
[56]
56. IgM antibody or IgM-like antibody according to claim 55, CHARACTERIZED by the fact that the antigen binding fragment is an scFv fragment.
[57]
57. IgM antibody or IgM-like antibody according to any one of claims 53 to 56, CHARACTERIZED by the fact that at least one heterologous polypeptide can specifically bind to CD3ε.
[58]
58. IgM antibody or IgM-like antibody according to claim 57, CHARACTERIZED by the fact that the J chain is a variant of the modified J chain of SEQ ID NO: 9 (V15J).
[59]
59. IgM antibody or IgM-like antibody according to claim 58, CHARACTERIZED by the fact that the modified J chain comprises the amino acid sequence of SEQ ID NO: 10 (V15J-Y102A).
[60]
60. IgM antibody or IgM-like antibody according to claim 58, CHARACTERIZED by the fact that the modified J chain comprises the amino acid sequence of SEQ ID NO: 23 (V15J-T103A).
[61]
61. IgM antibody or IgM-like antibody according to claim 58, CHARACTERIZED by the fact that the modified J chain comprises the amino acid sequence of SEQ ID NO: 24 (V15J-N49A).
[62]
62. Composition CHARACTERIZED by the fact that it comprises the IgM antibody or the IgM-like antibody according to any one of claims 1 to 61, and a pharmaceutically acceptable carrier.
[63]
63. Variant J chain or a functional fragment thereof, CHARACTERIZED by the fact that it comprises one or more substitutions, deletions or insertions of a single amino acid in relation to a reference J chain identical to the variant J chain, except for one or more substitutions or insertions single amino acid, where the variant J chain can affect the serum half-life of an IgM antibody or IgM-like antibody comprising the variant J chain.
[64]
64. Variant J chain according to claim 63, CHARACTERIZED by the fact that it comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 23, SEQ ID NO: 24 or any combination thereof.
[65]
65. Isolated polynucleotide CHARACTERIZED by the fact that it comprises a nucleic acid encoding an IgM or IgM-like subunit polypeptide according to any one of claims 1 to 61, wherein the subunit polypeptide comprises (a) a IgM or IgM-like heavy chain constant region or a multimerization fragment thereof, (b) an antibody light chain, or (c) a J chain, a modified J chain or fragment or functional variant thereof, or ( d) any combination thereof.
[66]
66. Polynucleotide according to claim 65, CHARACTERIZED by the fact that the subunit polypeptide comprises an IgM or IgM-like heavy chain constant region or a multimerization fragment thereof.
[67]
67. Polynucleotide according to claim 66, CHARACTERIZED by the fact that the subunit polypeptide comprises the amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 32 or SEQ ID NO: 34.
[68]
68. Polynucleotide according to claim 65, CHARACTERIZED by the fact that the subunit polypeptide comprises an antibody light chain.
[69]
69. Polynucleotide according to claim 65, CHARACTERIZED by the fact that the subunit polypeptide comprises a J chain, a modified J chain or any functional fragment or variant thereof.
[70]
70. Polynucleotide according to claim 69, CHARACTERIZED by the fact that the subunit comprises the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 or SEQ ID NO: 24.
[71]
71. Polynucleotide according to any one of claims 65 to 70, CHARACTERIZED by the fact that it comprises two, three or more nucleic acid sequences encoding two, three or more of the subunit polypeptides.
[72]
72. Expression vector, CHARACTERIZED by the fact that it comprises the polynucleotide according to any one of claims 65 to 71.
[73]
73. Host cell CHARACTERIZED by the fact that it comprises the polynucleotide, according to any one of claims 65 to 71, or the expression vector, according to claim 72.
[74]
74. Method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies comprising variant J chains, the method CHARACTERIZED by the fact that it comprises: (a) testing pentameric IgM antibodies or antibodies pentameric IgM-like cells comprising variant J chains or fragments thereof with regard to the increased serum half-life in an animal subject to a reference pentameric IgM antibody or reference pentameric IgM-like antibody, wherein the variant J chains or fragments thereof comprise inserts, defined amino acid deletions or substitutions, and wherein the reference pentameric IgM antibody or the pentameric IgM-like antibody comprises a J chain or a fragment thereof identical to the variant J chains, except for the defined amino acid insertions, deletions or substitutions; and (b) recovering the variant J chains or fragments thereof that confer increased serum half-life to the pentameric IgM antibodies or pentameric IgM-like antibodies to the reference pentameric IgM or antibody to the reference pentameric IgM.
[75]
75. Method for identifying variant J chains that can increase the serum half-life of pentameric IgM antibodies or pentameric IgM-like antibodies comprising the variant J chains, the method CHARACTERIZED by the fact that it comprises: (a) testing pentameric IgM antibodies or antibodies similar to pentameric IgMs comprising variant J chains or fragments thereof as to their level of binding to the Fc alpha-mu receptor (FcαμR), to the polymeric Ig receptor (pIgR) or to the FcαμR and pIgR, where the variant J chains or fragments thereof comprise defined amino acid insertions, deletions or substitutions; and (b) recovering the variant J chains or fragments thereof that confer reduced ability to bind to FcαµR, reduced ability to bind to pIgR or reduced ability to bind to FcαµR and pIgR in pentameric IgM antibodies or pentameric IgM-like antibodies with respect to a pentameric IgM antibody or a reference pentameric IgM-like antibody comprising a J chain or a fragment thereof identical to the variant J chains, except for the defined amino acid insertions, deletions or substitutions.
[76]
76. Method according to claim 75, CHARACTERIZED by the fact that it further comprises testing for pentameric IgM antibodies or pentameric IgM-like antibodies that include recovered variant J chains or fragments thereof for increasing the serum half-life in a subject animal with respect to a pentameric IgM antibody or a reference pentameric IgM-like antibody comprising a J chain or a fragment thereof identical to the recovered variant J chains or fragments thereof, except for the defined amino acid insertions, deletions or substitutions.
[77]
77. Method for identifying IgM variant heavy chain constant regions that can increase the serum half-life of an IgM antibody or IgM-like antibody comprising the IgM variant heavy chain constant regions, CHARACTERIZED by the fact that it comprises: (a ) test IgM antibodies or IgM-like antibodies comprising IgM heavy chain constant regions for increased serum half-life in an animal subject with respect to a pentameric IgM antibody or reference pentameric IgM-like antibody, where the constant regions of IgM variant heavy chain comprise defined amino acid insertions, deletions or substitutions and where the reference IgM antibody or IgM-like antibody comprises IgM heavy chain constant regions identical to variant IgM heavy chain constant regions, except for the inserts,
deletions or substitutions of defined amino acids; and (b) recovering the IgM antibodies or IgM-like antibodies comprising IgM variant heavy chain constant regions that impart increased serum half-life to IgM antibodies or IgM-like antibodies comprising the variant IgM heavy chain constant regions in reference to the IgM antibody or reference IgM-like antibody.
[78]
78. Method for identifying IgM variant heavy chain constant regions that may increase the serum half-life of an IgM antibody or IgM-like antibody comprising the IgM variant heavy chain constant regions, CHARACTERIZED by the fact that it comprises: (a ) test IgM antibodies or IgM-like antibodies comprising IgM heavy chain constant regions variant for their level of binding to the Fc alpha-mu receptor (FcαμR), Fc mu receptor (FcµR), polymeric Ig receptor (pIgR), any combination of two of the receptors or all three receptors, wherein the IgM variant heavy chain constant regions comprise defined amino acid insertions, deletions or substitutions; and (b) recovering IgM antibodies or IgM-like antibodies comprising variant IgM heavy chain constant regions that confer reduced FcαµR binding capacity, reduced FcµR binding capacity, reduced pIgR binding capacity, binding capacity to any one of the two receptors reduced or reduced binding capacity to all three receptors, in IgM antibodies or IgM-like antibodies comprising the IgM heavy chain constant regions variant with respect to a reference IgM antibody or IgM-like antibody comprising constant regions IgM heavy chain identical to the variant IgM heavy chain constant regions, except for the defined amino acid insertions, deletions or substitutions.
[79]
79. Method according to claim 78, CHARACTERIZED by the fact that it further comprises testing for recovered IgM antibodies or IgM-like antibodies comprising constant regions of IgM heavy chain variant with regard to increasing serum half-life in an animal subject in with respect to an IgM antibody or reference IgM-like antibody comprising variant IgM heavy chain constant regions identical to variant IgM heavy chain constant regions, except for defined amino acid insertions, deletions or substitutions.

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法律状态:
2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201862637186P| true| 2018-03-01|2018-03-01|

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US62/637,186|2018-03-01|

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PCT/US2019/020374|WO2019169314A1|2018-03-01|2019-03-01|IgM Fc AND J-CHAIN MUTATIONS THAT AFFECT IgM SERUM HALF-LIFE|

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