Humanized antibodies against CD79b

Abstract

The present invention relates to a humanized antibody or a target binding fragment or derivative thereof that retains target binding capacity, which binds to human CD79b.

Description

Technical Field

[0001] The present application relates to humanized antibodies against CD79b. Background Art

[0002] Despite significant progress in recent years, many cancer types remain difficult or even impossible to successfully treat. This is particularly true for B-cell-related malignancies. Targeted therapies already exist for targets such as CD20, CD19, and CD22.

[0003] CD79b (immunoglobulin-associated β chain) is also an antigen on B cells. Studies have shown that it may have advantages over CD20 and others as a target.

[0004] CD79b, together with CD79a and surface immunoglobulin, forms the B cell receptor (BCR), which is expressed in more than 90% of B cell non-Hodgkin lymphoma (NHL) malignancies. CD79b can be internalized upon antibody binding. Due to this functional property, CD79b has the potential to selectively deliver target molecules to B cells in non-Hodgkin lymphoma (NHL).

[0005] Studies have shown that anti-CD79b antibodies are able to block B cell proliferation induced through the B cell receptor, CD40, CD180, and chondroitin sulfate, but not through TLR4 or TLR9 (Brühl et al., 2015).

[0006] Anti-CD20 antibody therapy remains the standard of care for many patients with B-cell malignancies. About two-thirds of patients benefit from the therapy, but that means one-third do not.

[0007] In this context, CD79b appears to offer a new treatment option. According to a phase III clinical trial reported by Tilly et al. (2021) involving 879 patients with diffuse large B-cell lymphoma aged between 18 and 80 years, these patients had a medium-to-high risk of poor prognosis. Patients received standard treatment with anti-CD20 antibody combined with chemotherapy (R-CHOP) or antibody-drug conjugate combined with chemotherapy of anti-CD79b antibody (pola-R-CHP). Significantly more patients in the pola-R-CHP group survived without disease progression. Patients treated with antibody-drug conjugate combined with chemotherapy had a significant reduction in the risk of disease progression, relapse, and death from lymphoma.

[0008] The antibody-drug conjugate used in this study was Polatuzumab vedotin Also known as DCDS4501A or RG7596.

[0009] Polivy is an antibody-drug conjugate comprising an anti-CD79b antibody of IgG structure and a toxin MMAE coupled thereto via a linker / spacer. The linker / spacer consists of: p-aminobenzyl carbamate coupled to the free thiol group of the cysteine ​​residue in the cysteine ​​engineered antibody ("Thiomab"); a cathepsin-cleavable linker comprising citrulline and valine; and an attachment group consisting of hexanoic acid and maleimide to which the toxin is coupled. The average drug-antibody ratio (DAR) of Polivy is 3.5 MMAE molecules per antibody, so there is variability in DAR.

[0010] Polatuzumab is a humanized variant of the murine anti-CD79b antibody SN8, which was first described by Okazaki et al. in 1993. Polatuzumab and Polatuzumab vedotin are disclosed in patents such as EP2176296 and US8545850.

[0011] It is an object of the present invention to provide new and improved cancer treatment options.

[0012] These and other objects are achieved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred in certain circumstances. Likewise, the description discloses further embodiments of the invention which may be preferred in certain circumstances. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 , Figure 2 and Figure 3 The results of in vitro cell activity assays of four recombinant immunotoxins according to the present invention (IgG antibodies bound to the protein toxin anisoplin via a G4S linker) and a benchmark antibody-drug conjugate (Polatuzumab Vedotin) are shown. It can be seen that the IC50 (in vitro potency) of the four immunotoxins of the present invention is significantly better than that of the benchmark antibody-drug conjugate.

[0014] Figure 4 and Figure 5 The results of size exclusion chromatography experiments of a recombinant immunotoxin according to the present invention (IgG antibody conjugated to the protein toxin anisoplin via a G4S linker) and a benchmark recombinant immunotoxin (Polatuzumab conjugated to the protein toxin anisoplin via a G4S linker) are shown. Figure 4 A: ATB 704, Figure 4 B:ATB-580, Figure 5 A: Column calibration, Figure 5B: Benchmark (Polatuzumab). The recombinant immunotoxin according to the invention is highly stable, whereas the Polatuzumab-based benchmark (ATB-452) shows a higher tendency to aggregate.

[0015] Figure 6 and Figure 7 Results of immunohistochemical (IHC) staining of frozen human tissues from three donors under conditions that favor high-affinity interaction with the cognate receptor (on-target binding) are shown. Specific staining was observed only in CD79b-positive human tissues (e.g., lymph nodes, spleen, and thymus) for the recombinant immunotoxins ATB-580, ATB-693, ATB-697, and ATB-704 (IgG antibodies conjugated to the protein toxin anisoplin via a G4S linker). No off-target binding to CD79b-negative tissues was observed for all tested compounds.

[0016] Figure 8 The in vivo antitumor activity of four recombinant immunotoxins according to the invention (IgG antibodies conjugated to the protein toxin anisoplin via a G4S linker) is compared to a benchmark recombinant immunotoxin (Polatuzumab conjugated to the protein toxin anisoplin via a G4S linker, referred to herein as "ATB-747"). For this purpose, a subcutaneous CDX model in SCID mice (transplanted with a representative cell line of B-NHL) was used.

[0017] Fig. 9 A sequence alignment of different CD79B isoforms is shown, with epitopes discussed in the specification marked in bold. DETAILED DESCRIPTION

[0018] According to one aspect of the present invention, an antibody or a target binding fragment or derivative thereof that retains the ability to bind to human CD79b is provided.

[0019] a) comprising a set of six heavy chain / light chain complementarity determining regions (CDRs) contained in a heavy chain / light chain variable region sequence pair selected from any one of the following SEQ ID NO pairs:

[0020] SEQ ID NO: 3 and 4,

[0021] SEQ ID NO: 13 and 14,

[0022] SEQ ID NO: 23 and 24, or

[0023] SEQ ID NO: 33 and 34,

[0024] b) comprising a set of six heavy chain / light chain complementarity determining regions (CDRs) selected from the group consisting of: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, in the order:

[0025] i) SEQ ID NO: 5, 6, 7, 8, 9 and 10,

[0026] ii) SEQ ID NO: 15, 16, 17, 18, 19 and 20,

[0027] iii) SEQ ID NO: 25, 26, 27, 28, 29 and 30, or

[0028] iv) SEQ ID NO: 35, 36, 37, 38, 39 and 40,

[0029] c) it comprises a set of heavy chain / light chain complementarity determining regions (CDRs) as described in option b), and at least one of the CDRs has a maximum of 3 amino acid substitutions relative to the CDR contained in the corresponding SEQ ID NO, and / or

[0030] d) it comprises a set of heavy chain / light chain complementarity determining regions (CDRs) as described in option b) or c), and at least one of the CDRs has a sequence identity of ≥ 66% with the CDR contained in the corresponding SEQ ID NO.

[0031] The CDRs are embedded in a suitable protein framework so as to be able to bind to human CD79b. The following table lists the antibodies according to the invention in a simplified form.

[0032] Table 1: Antibody names and sequences used in this article

[0033]

[0034] In one embodiment, the antibody is a humanized antibody or fragment thereof.

[0035] Methods for the production and / or selection of humanized monoclonal antibodies are known in the prior art. For example, US6331415 to Genentech describes the production of chimeric antibodies, US6548640 to the Medical Research Council describes CDR grafting technology, and US5859205 to Celltech describes the production of humanized antibodies.

[0036] Humanized antibodies are antibodies whose complementary determining regions (CDRs) are derived from a parent antibody of a non-human species and transplanted into the framework (at least the variable region) of a human antibody (e.g., IgG1, IgG2, or IgG4). Humanized antibodies bind to the same target as the parent antibody, but because they are transplanted into a human framework, they have lower immunogenicity (e.g., HAMA response). Therefore, humanized antibodies are structurally different from their parent (e.g., mouse) antibodies.

[0037] During the humanization process, after the CDRs are grafted onto the human framework, an affinity maturation step is usually performed to regain the affinity lost during the grafting process. This process further modifies the sequence of the human antibody, including its CDRs.

[0038] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found in the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al. (1977), Kabat et al. (1991), and Chothia et al. (1987), wherein these definitions may include overlapping or subset amino acid residues when compared to each other.

[0039] Nonetheless, application of either definition to refer to a CDR of an antibody, grafted antibody, or variants thereof is within the scope of the term as defined and used herein.

[0040] Preferably, the CDRs described in this specification are identified according to the Kabat or Chothia numbering system listed in Table 2.

[0041] Table 2: CDR Definition

[0042] Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-65 53-55 VH CDR3 95-102 96-101 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0043] As used herein, the term "framework" when referring to an antibody variable region refers to all amino acid residues within the antibody variable region excluding the CDR regions.

[0044] Thus, the variable region framework is approximately 100-120 amino acids in length, but refers only to those amino acids outside the CDRs.

[0045] In one embodiment, the term "capable of binding to target X" is understood to mean that the corresponding binding domain is -4 or smaller K D Value binding target. K D is the equilibrium dissociation constant, which is the k between the antibody or its fragment and the antigen off / k on Ratio. K D The value is inversely proportional to the affinity. D The value is related to the concentration of the antibody or fragment (the amount of antibody or fragment required for a particular experiment), so KD The lower the value (lower the concentration), the higher the affinity of the binding domain. The following table shows typical K values ​​for monoclonal antibodies. D Value range:

[0046] Table 3: K D Value and molar value

[0047] <![CDATA[K D Value Range]]> Molar Range <![CDATA[10 -4 Up to 10 -6 ]]> Micromolar (μM) <![CDATA[10 -7 Up to 10 -9 ]]> Nanomolar (nM) <![CDATA[10 -10 Up to 10 -12 ]]> Picomole (pM) <![CDATA[10 -13 Up to 10 -15 ]]> Femtomole (fM)

[0048] Preferably, the antibody or fragment thereof has at most 2 amino acid substitutions, more preferably at most 1 amino acid substitution.

[0049] Preferably, at least one CDR of the antibody or fragment thereof has a sequence identity with the corresponding SEQ ID NO of ≥67%; ≥68%; ≥69%; ≥70%; ≥71%; ≥72%; ≥73%; ≥74%; ≥75%; ≥76%; ≥77%; ≥78%; ≥79%; ≥80%; ≥81%; ≥82%; ≥83%; ≥84%; ≥85%; ≥86%; ≥87%; ≥88%; ≥89%; ≥90%; ≥91%; ≥92%; ≥93%; ≥94%; ≥95%; ≥96%; ≥97%; ≥98%; ≥99%; and most preferably 100%.

[0050] As used herein, "sequence identity percentage" is determined by comparing two optimally aligned biological sequences (amino acid sequences or polynucleotide sequences) within a comparison window, wherein portions of the corresponding sequences in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence, while the reference sequence does not contain additions or deletions to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0051] The term "identical" or percentage "identity" when referring to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical sequences. If two sequences have a specific percentage of identical amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) within a specific region (when no region is specified, then within the scope of the entire reference sequence) after maximum correspondence alignment, the two sequences are "substantially identical". The above-mentioned maximum correspondence alignment is performed within a comparison window or specified region by one of the following sequence comparison algorithms or by manual alignment and visual inspection. The present disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. For amino acid sequences, identity or substantial identity can exist over a region of at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the entire length of the reference sequence. For shorter amino acid sequences (e.g., sequences of 20 or fewer amino acids), substantial identity exists when one or two amino acid residues are conservatively substituted as defined herein.

[0052] Preferably, at least one of the CDRs is subjected to CDR sequence modification, comprising:

[0053] · Mature affinity;

[0054] Reduced immunogenicity.

[0055] Affinity maturation refers to a method of improving the affinity of a given antibody by an in vitro process. Similar to the natural counterpart, in vitro affinity maturation is based on the principle of mutation and selection. It has been successfully used to optimize antibodies, antibody fragments or other peptide molecules (such as antibody mimetics). Random mutations are introduced into the CDR by radiation, chemical mutagens or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two to three rounds of mutation and selection using display methods such as phage display usually produce antibody fragments with affinity in the low nanomolar range. For the principle, see Eylenstein et al. (2016) or US20050169925A1, the contents of which are incorporated herein by reference for the purpose of feasibility.

[0056] The engineered antibodies contain CDR regions derived from the mouse sequence, which, along with any necessary framework back-mutations, have been transplanted into the sequence-derived V regions. Therefore, when the humanized antibodies are administered to patients, the CDRs themselves may cause an immunogenic response. Methods for reducing the immunogenicity caused by CDRs are disclosed in Harding et al. (2010) or US2014227251A1, the contents of which are incorporated herein by reference for the purpose of implementation.

[0057] According to one embodiment of the present invention, the antibody or fragment thereof comprises

[0058] a) a heavy chain / light chain variable region (HCVD / LCVD) pair listed in any of the following SEQ ID NO pairs:

[0059] SEQ ID NO: 3 and 4,

[0060] SEQ ID NO: 13 and 14,

[0061] SEQ ID NO: 23 and 24, or

[0062] SEQ ID NO: 33 and 34,

[0063] b) a heavy chain / light chain variable region (HCVD / LCVD) pair as described in a), and satisfying

[0064] The heavy chain variable region (HCVD) has the same sequence as the HCVD contained in the corresponding SEQ ID NO.

[0065] ≥ 80% sequence identity, and / or

[0066] The light chain variable region (LCVD) has the same sequence as the LCVD contained in the corresponding SEQ ID NO.

[0067] ≥80% sequence identity,

[0068] c) The heavy chain / light chain variable region (HCVD / LCVD) pair as described in option a) or b), at least one HCVD or LCVD has a maximum of 10 amino acid substitutions relative to the HCVD or LCVD contained in the corresponding SEQ ID NO.

[0069] The prerequisite is that the antibody or fragment thereof is capable of binding to human CD79b.

[0070] "Variable region" when referring to an antibody or its heavy or light chain, refers to the portion of the molecule that confers antigen binding ability and is not part of the constant region. The term is intended to include functional fragments that retain all or part of the intact variable region binding function. Variable region binding fragments include, for example, Fab, F(ab)2, Fv, single-chain Fv (scFv) and other functional fragments. These functional fragments are well known to those skilled in the art. Therefore, the use of these terms when describing functional fragments of heterologous variable regions is intended to correspond to definitions well known to those skilled in the art. These terms are described, for example, in Huston et al. (1993) or Plückthun and Skerra (1990).

[0071] Preferably, the sequence identity of the heavy chain variable region (HCVD) and / or the light chain variable region (LCVD) to the corresponding SEQ ID NO is:

[0072] ≥81%; ≥82%; ≥83%; ≥84%; ≥85%; ≥86%; ≥87%; ≥88%; ≥89%; ≥90%; ≥91%; ≥92%; ≥93%; ≥94%; ≥95%; ≥96%; ≥97%; ≥98%; ≥99%; most preferably 100%.

[0073] According to one embodiment of the invention, at least one amino acid substitution is a conservative amino acid substitution.

[0074] As used herein, a "conservative amino acid substitution" has less effect on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are generally divided into six categories based on their structure and the general chemical properties of the R group.

[0075] In certain embodiments, "conservative amino acid substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having the following side chains:

[0076] Basic side chains (e.g. lysine, arginine, histidine),

[0077] Acidic side chains (e.g. aspartic acid, glutamic acid),

[0078] Uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine

[0079] acid, threonine, tyrosine, cysteine),

[0080] Nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

[0081] β-branched side chains (e.g., threonine, valine, isoleucine), and

[0082] • Aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

[0083] Other conservative amino acid substitutions may also occur between amino acid side chain families, such as replacing aspartic acid with asparagine to change the charge of the peptide. Conservative substitutions can also include substitutions of chemically homologous non-natural amino acids (e.g., replacing leucine with a synthetic non-natural hydrophobic amino acid, or replacing tryptophan with a synthetic non-natural aromatic amino acid).

[0084] According to one embodiment of the present invention, the human CD70b bound by the antibody or fragment thereof comprises:

[0085] a) an amino acid sequence as shown in any one of SEQ ID NOs: 41-44, or

[0086] b) an amino acid sequence having at least 80% sequence identity to SEQ ID NO:41-44, provided that the sequence retains CD79b activity.

[0087] In some embodiments, human CD79b comprises an amino acid sequence having ≥81%, preferably ≥82%, more preferably ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or most preferably ≥99% sequence identity to SEQ ID NO:41-44.

[0088] SEQ ID NO:41 represents the amino acid sequence of human CD79b, which can be obtained through NCBI reference. In general, there are different CD79b variants and isomers, which are disclosed herein as SEQ ID NOs:42-44. Similarly, mutants containing conservative or silent amino acid substitutions exist or may exist, and these mutants retain all or at least most of the CD79b activity. These isomers, variants and mutants are included in the above-mentioned identity range, except for dysfunctional or inactive variants and mutants.

[0089] In this context, the inventors unexpectedly found that the antibodies according to the present invention are able to bind to all four CD79b isoforms mentioned above. This finding is surprising because the epitope ARSEDRYRNPKGSACSRIWQS (SEQ ID NO: 61) recognized by existing anti-CD79b antibodies (such as SN8) is only present in isoforms 1 and 3, but missing in isoforms 2 and 4 (see Fig. 9Based on this, the antibodies of the present invention have an unexpectedly broad target spectrum.

[0090] According to one embodiment of the present invention, the antibody or fragment thereof is a monoclonal antibody, or a target binding fragment or derivative thereof that retains the ability to bind to human CD79b.

[0091] According to one embodiment of the present invention, the antibody or fragment thereof is at least one selected from the group consisting of: IgG, scFv, Fab or (Fab)2.

[0092] The term "monoclonal antibody (mAb)" as used herein refers to an antibody composition having a homogeneous antibody population, ie, a homogeneous population consisting of intact immunoglobulins or fragments or derivatives thereof that retain target binding ability.

[0093] It is particularly preferred that such antibodies are IgG antibodies or fragments or derivatives thereof that retain target binding ability. Immunoglobulin G (IgG) is a type of antibody. IgG accounts for approximately 75% of human serum antibodies and is the most common type of antibody circulating in the blood. IgG molecules are produced and released by plasma cells B cells. Each IgG molecule has two antigen binding sites.

[0094] IgG antibodies are large molecules with a molecular weight of about 150kDa and are composed of four peptide chains. It contains two identical γ-type heavy chains (about 50kDa) and two identical light chains (about 25kDa), so it has a quaternary structure of a tetramer. The two heavy chains are connected to each other by disulfide bonds and are each connected to a light chain. The resulting tetramer has two identical halves that together form a Y-shaped structure. Each forked end contains an identical antigen binding site. The Fc region of IgG has a highly conserved N-glycosylation site. The N-glycans attached to this site are mainly complex biantenna structures with core fucosylation. In addition, a small number of these N-glycans also carry bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0095] According to one embodiment of the present invention, the antibody is in at least one form selected from the group consisting of IgG1, IgG2 or IgG4.

[0096] As used herein, the term "fragment" shall refer to fragments of such antibodies that retain target binding ability, for example:

[0097] CDR (complementarity determining region)

[0098] Highly variable region,

[0099] Variable region (Fv)

[0100] IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions)

[0101] IgG or IgM light chain (consisting of the VL and CL regions), and / or

[0102] Fab and / or F(ab)2.

[0103] As used herein, the term "derivative" shall refer to a protein construct that is structurally different from the common antibody concept but still has certain structural relationships, such as scFv, Fab and / or F(ab)2, and a bispecific, trispecific or higher specific antibody construct, and further retains target binding ability. These items will be explained below.

[0104] Other antibody derivatives known to those skilled in the art include bispecific antibodies (Diabodies), camelid antibodies (Camelid Antibodies), nanobodies (Nanobodies), domain antibodies (Domain Antibodies), bivalent homodimers with two chains consisting of scFv, IgA (two IgG structures connected by J chain and secretory component), shark antibodies, antibodies consisting of New World Primate Framework plus non-New World Primate CDR, dimerization constructs comprising CH3+VL+VH, and antibody conjugates (e.g., antibodies or fragments or derivatives thereof connected to toxins, cytokines, radioisotopes or labels). These types are described in detail in the literature and can be used by those skilled in the art based on the present disclosure without additional creative activities.

[0105] & Milstein (1975) disclosed a method for producing hybridoma cells.

[0106] Methods for producing and / or screening fully human monoclonal antibodies are known in the art. These methods may involve the use of transgenic animals immunized with the corresponding protein or polypeptide, or the use of suitable display technologies, such as yeast display, phage display, B cell display or ribosome display, to screen antibodies against human CD79b from libraries in a fixed phase.

[0107] In vitro antibody libraries are disclosed in US6300064 to MorphoSys and US6248516 to MRC / Scripps / Stratagene. Phage display technology is disclosed, for example, in US5223409 to Dyax. Transgenic mammalian platforms are described, for example, in EP1480515A2 to TaconicArtemis.

[0108] IgG, IgM, scFv, Fab and / or F(ab)2 are antibody forms well known to those skilled in the art. Related techniques can be obtained from corresponding textbooks.

[0109] As used herein, the term "Fab" relates to an IgG / IgM fragment containing an antigen binding region, which fragment consists of one constant region and one variable region each of the antibody heavy and light chains.

[0110] As used herein, the term "F(ab)2" relates to an IgG / IgM fragment consisting of two Fab fragments linked by a disulfide bond.

[0111] As used herein, the term "scFv" refers to a single-chain variable fragment, which is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together by a short linker, usually serine (S) or glycine (G). Despite the removal of the constant region and the introduction of a linker peptide, this chimeric molecule retains the specificity of the original immunoglobulin.

[0112] Modified antibody forms include, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, etc. These types are described in detail in the literature, and those skilled in the art can use them based on the present disclosure and increase creative activities.

[0113] In one or more embodiments, the antibody or fragment thereof is an isolated antibody, or a target binding fragment or derivative that retains target binding ability, or an isolated antibody mimetic.

[0114] In one or more embodiments, the antibody is an engineered or recombinant antibody, or a target binding fragment or derivative that retains target binding ability, or an engineered or recombinant antibody mimetic.

[0115] According to one embodiment of the present invention, the antibody or fragment thereof is an antibody selected from at least one of the following groups: IgG, scFv, Fab or (Fab)2.

[0116] According to another aspect of the present invention, a nucleic acid encoding at least one chain of the binding agent described above is provided.

[0117] In one embodiment, a nucleic acid or a set of nucleic acid pairs is provided, which encode the heavy chain and light chain of a binding agent, respectively, and the binding agent is a monoclonal antibody having a heterologous structure of at least one light chain and one heavy chain.

[0118] Such nucleic acids can be used to recombinantly produce the antibody or fragment or derivative thereof in a suitable expression system (eg, CHO cells or tobacco cells).

[0119] Such nucleic acids can also be used for pharmaceutical purposes. The nucleic acid can be an RNA molecule, or an RNA derivative comprising a modified nucleotide (such as pseudouridine (Ψ) or N-1 methyl pseudouridine (m1Ψ)), to provide stability and reduce immunogenicity (see, for example, US8278036 and US9428535, the contents of which are incorporated herein for implementation purposes). In another embodiment, RNA comprising the richest GC codons is selected to provide stability and reduce immunogenicity (see, for example, EP1392341, the contents of which are incorporated herein for implementation purposes). For example, the mRNA can be delivered in a suitable liposome and comprise a specific sequence or modified uridine nucleoside to avoid immune response and / or improve folding and translation efficiency, sometimes comprising a cap modification at the 5' end and / or 3' end to target it to a specific cell type.

[0120] The nucleic acid can also be a DNA molecule. In this case, the molecule can be a cDNA, which is optionally integrated into a suitable vector, such as an attenuated non-pathogenic virus, or provided as one or more plasmids. Such plasmids can be administered to patients, for example, by an electroporation device (such as the device disclosed in patent EP3397337B1), the contents of which are incorporated herein for implementation purposes.

[0121] Typically, due to the degeneracy of the genetic code, there are a large number of different nucleic acids that can encode such chains. Those skilled in the art are fully capable of determining whether a given nucleic acid meets the above criteria. On the other hand, those skilled in the art are fully capable of reverse designing a suitable encoding nucleic acid from a given amino acid sequence based on a codon usage table. To this end, software tools such as "reverse translate" (https: / / www.bioinformatics.org / sms2 / rev_trans.html) provided by the online tool "sequence manipulation suite" can be used. Therefore, there are a large number of alternative DNA and RNA sequences encoding the claimed protein sequence. These alternative sequences should be considered to fall within the scope of the present invention.

[0122] According to another aspect of the present invention, a recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide conjugate is provided. This embodiment comprises the antibody or fragment thereof described above.

[0123] As used herein, the term "recombinant immunotoxin" refers to a fusion construct comprising at least (i) an antibody or fragment thereof as described above, and (ii) a protein toxin or protoxin fused thereto. Such recombinant immunotoxins can be produced in a suitable recombinant expression system without the need for subsequent conjugation of the toxin to the antibody or fragment thereof.

[0124] As used herein, the term "protein toxin" or "protein protoxin" is intended to encompass cytotoxic and / or cytostatic proteins or precursor forms thereof.

[0125] As used herein, the term "cytostatic protein" refers to a protein that is capable of inhibiting cell proliferation or cell division but not necessarily killing the cell. Suitably, the cytostatic agent inhibits the proliferation of tumor cells.

[0126] As used herein, the term "cytotoxic protein" refers to a protein that is harmful to cells and ultimately causes cell death. In some embodiments, the cytotoxic protein causes damage to rapidly dividing cells (such as tumor cells) and causes tumor cell death, especially causing tumor cell death without causing damage to non-tumor cells or causing less damage.

[0127] The term "protein toxin" or "protein protoxin" refers, without limitation, to toxins that are chemically proteins (i.e., peptides ≥50 amino acid residues in length) or polypeptides (i.e., peptides ≥10 to ≤50 amino acid residues in length). In the meaning of the present invention, a protoxin is a precursor of a toxin, also called a latent toxin, which needs to be activated by, for example, cleavage of an inhibitory amino acid sequence or undergoing a conformational change. The terms "protoxin" and "protein protoxin" are used interchangeably herein and refer to the same subject matter.

[0128] Such protein toxins or protoxins may be selected from the group consisting of ribotoxins, endoribonucleases (RNases), ribosome inactivating proteins (RIPs) and AB toxins.

[0129] As used herein, the term "ribotoxin" refers to a group of extracellular ribonucleases secreted by fungi. The most notable feature of ribotoxins is their extremely high specificity. They inactivate ribosomes by cleaving a single phosphodiester bond in a universally conserved sequence in rRNA. This cleavage leads to cell death through apoptosis. However, since they are extracellular proteins, they must first enter the cells that constitute their target in order to exert their cytotoxic effects. This entry constitutes a step that determines the speed of their action.

[0130] Ribotoxins have been detected in many different fungi, including insect pathogenic fungi and edible fungi, but the three-dimensional structures of only three of them have been resolved: α-sarcin (SEQ ID NO:56, and its deimmunized variant SEQ ID NO:57), restrictocin, and Hirsutellin A (HtA, SEQ ID NO:48). The first two are produced by Aspergillus giganteus and Aspergillus restrictus, respectively, and are almost identical. HtA, produced by the insect pathogenic fungus Hirsutella thompsonii, is much smaller and shows only 25% sequence identity with other larger ribotoxins. Despite this, it retains all the functional characteristics of this family. A second ribotoxin similar to HtA is anisoplin (SEQ ID NO: 49, and its analogs and deimmunized variants, disclosed herein as SEQ ID NOs: 50, 52, 53, and 54), which is known to have 70% sequence identity with HtA. It is produced by Metarhizium anisopliae, another insect pathogenic fungus. Other ribotoxins useful in the present invention include Angiogenin (SEQ ID NO: 51) and Ageritin (SEQ ID NO: 55).

[0131] As used herein, the term "RNase" refers to a group of nucleases ("ribonucleases") that catalyze the degradation of RNA into smaller components. In the meaning of the present invention, ribonucleases act as endoribonucleases. In some embodiments, the RNase is selected from the following group:

[0132] Onconase (rampirinase, frog RNase): There are different Onconase variants, examples of which are published in UniProt identifiers Q8UVX5, Q9I8V8, Q6EUW9,

[0133] Although some examples in this application use Q8UVX5, other Onconase variants can also be used.

[0134] RNase 1: Pancreatic ribonuclease (e.g. hRNase1, e.g. UniProt identifier

[0135] P07998 (SEQ ID NO: 58)).

[0136] RNase 5: angiopoietin (e.g. hRNase 5, e.g. UniProt identifier

[0137] P03950).

[0138] • RNase 2: non-secreted ribonuclease (eg hRNase2, eg UniProt identifier P10153).

[0139] ·RNase 3: Eosinophil cationic protein (e.g. hRNase3 / Drosha, e.g.

[0140] UniProt identifiers Q9NRR4 or P12724).

[0141] RNase 4: Ribonuclease 4 (e.g. hRNase4, e.g. UniProt identifier

[0142] P34096).

[0143] RNase 6: RNase K6 / RNase T2 / RNase K3 (e.g.

[0144] hRNase6, e.g. UniProt identifier Q93091).

[0145] ·RNase 7: Ribonuclease 7 / ribonuclease A E1 (e.g. hRNase7, e.g.

[0146] UniProt identifier Q9H1E1).

[0147] RNase 8: Ribonuclease 8 (e.g. hRNase8, e.g. UniProt identifier

[0148] Q8TDE3).

[0149] The above UniProt identifiers are for exemplary purposes only. Other variants can also be used. Those skilled in the art can find such variants in the corresponding databases through routine efforts.

[0150] Ribosome inactivating proteins (RIPs) are toxic N-glycosidases that depurinate eukaryotic and prokaryotic rRNA, thereby preventing protein synthesis during translation. RIPs are widely found in various plant species and different tissues. These proteins are known to play a key role in defense against pathogens and are thought to confer disease resistance. To date, plant-derived RIPs have been found in more than 50 different plant species from 14 families, including Cucurbitaceae, Euphorbiaceae, Poaceae, and Caryophyllales. In addition to plants, RIPs have also been found in bacteria, fungi, algae, and even mosquitoes.

[0151] RIPs constitute a large family of proteins that can be classified based on their structural composition, namely, RIP type I and type II.

[0152] The molecular weight of type I RIP is low, about 30 kDa, and it is a single-chain protein. The single chain of type I RIP consists of an enzymatic active domain (A domain or α domain) with N-glycosidase activity.

[0153] Type II RIPs are larger proteins with a molecular weight between 50 and 65 kDa, characterized by an enzymatically active A chain and a slightly larger B chain (or β chain, a lectin subunit) with a galactose-like glycosyl group.

[0154] In addition to type I and II RIPs, a third class, type III RIPs, has been reported, which has fewer members and possesses an N-terminal domain associated with the A domain of RIP and is bound to a C-terminal domain of unknown function.

[0155] According to various embodiments, the ribosome inactivating protein (RIP) is selected from at least one of the group consisting of:

[0156] Momordin

[0157] Bryodin I (SEQ ID NO: 62)

[0158] · Cucurmosin

[0159] Bryodin II (SEQ ID NO: 63)

[0160] Trichosanthin

[0161] ·Karasurin

[0162] ·MOMC

[0163] ME1, and / or

[0164] ME2

[0165] AB toxins are two-component protein complexes secreted by a variety of pathogenic bacteria. They can be classified as type III toxins because they interfere with intracellular functions. They are called AB toxins because of their components: the "A" component is usually the "active" part, while the "B" component is usually the "binding" part. The "A" subunit has enzymatic activity and is translocated into the host cell after a conformational change in the membrane-bound transporter "B" subunit. These proteins consist of two separate polypeptide chains, corresponding to the A / B subunit parts. The enzyme component (A) enters the cell through endosomes generated by the oligomeric binding / transporter protein (B) and prevents actin polymerization by ADP-ribosylation of monomeric G-actin.

[0166] Examples of the "A" component of AB toxins include C. perfringens iota toxin Ia, C. botulinum C2 toxin CI, and Clostridium difficile ADP-ribosyltransferase. Other homologous proteins are also found in Clostridium spiroforme.

[0167] An example of the B component of an AB toxin is the Bacillus anthracis Protective Antigen (PA) protein. Bacillus anthracis secretes three toxin factors: Protective Antigen (PA), Edema Factor (EF), and Lethal Factor (LF). Each is a heat-labile protein of approximately 80 kDa. PA forms the "B" portion of the exotoxin and allows the "A" portion (consisting of either EF or LF) to enter the target cell. The PA protein constitutes the core portion of the complete anthrax toxin and transports the A portion into the host cell after being assembled into a heptamer in the membrane.

[0168] Diphtheria toxin is also an AB toxin. It inhibits protein synthesis in host cells by phosphorylating eukaryotic elongation factor 2, an essential component of protein synthesis. Exotoxin A of Pseudomonas aeruginosa is another AB toxin that targets eukaryotic elongation factor 2.

[0169] AB5 toxins are often considered a type of AB toxin, characterized by the presence of a B pentamer. Less commonly, the term "AB toxin" is used to emphasize the monomeric nature of the B component.

[0170] The two-stage mechanism of action of AB toxins is of particular interest in cancer therapy research. The general idea is to modify the B component of an existing toxin so that it selectively binds to malignant cells. This approach combines the results of cancer immunotherapy with the high toxicity of AB toxins, resulting in a new class of chimeric protein drugs called immunotoxins.

[0171] In one embodiment, the recombinant immunotoxin comprises

[0172] i) an antibody according to the above description,

[0173] ii) a toxin as described above, and

[0174] iii) a peptide linker connecting the two components i) and ii) above.

[0175] Linkers suitable for this purpose are disclosed herein as SEQ ID NOs: 45, 46, 47 and 60.

[0176] In one embodiment, the recombinant immunotoxin comprises antibody 580, which is defined by the amino acid sequence of SEQ ID NOs: 3-10 (SEQ ID NOs: 3 and 4: variable regions, SEQ ID NOs: 5-10: CDRs).

[0177] In one embodiment, the recombinant immunotoxin comprises antibody 704, which is defined by the amino acid sequence SEQ ID NOs: 33-40 (SEQ ID NOs: 33 and 34: variable region, SEQ ID NOs: 35-40: CDRs). In one embodiment, the recombinant immunotoxin comprises a ribotoxin anisoplin or an analog thereof (selected from any one of SEQ ID NOs: 49, 50, 52-54). In one embodiment, the recombinant immunotoxin comprises a peptide linker that cannot be cleaved by a mammalian protease, such as a G4S linker (SEQ ID NO: 60).

[0178] In one embodiment, the recombinant immunotoxin comprises

[0179] (i) antibody 580 defined by the amino acid sequence of SEQ ID NOs: 3-10,

[0180] (ii) anisoplin, a ribotoxin having the sequence SEQ ID NO: 49, and

[0181] (iii) a peptide linker having the sequence SEQ ID NO:60.

[0182] In one embodiment, the recombinant immunotoxin comprises

[0183] (i) antibody 704 defined by the amino acid sequence of SEQ ID NOs: 33-40,

[0184] (ii) anisoplin, a ribotoxin having the sequence SEQ ID NO: 49, and

[0185] (iii) a peptide linker having the sequence SEQ ID NO:60.

[0186] Preferably, the antibody in the recombinant immunotoxin is in the IgG1 form.

[0187] As used herein, the term "immunocytokine" refers to a fusion construct comprising at least (i) an antibody or fragment thereof as described above, and (ii) an immunomodulatory cytokine fused thereto. Such recombinant immunocytokines can be produced in a suitable recombinant expression system without the need for subsequent coupling of the cytokine to the antibody or fragment thereof.

[0188] Immunocytokines can be used to improve site-specific delivery and extend the half-life of cytokines. Immunocytokines are delivered systemically but can be targeted via specific tumor antigens. Cytokines suitable for fusion with antibodies include, but are not limited to, TNFα, IL2, IL12, and IL15. In this way, the maximum tolerated dose can be increased, for example, the maximum tolerated dose of IL12 was found to be 30 times higher than when IL12 was used alone.

[0189] As used herein, the term "antibody drug conjugate" refers to a construct comprising an antibody or fragment thereof covalently bound to a toxin. The toxin is typically a small molecule toxin with a molecular weight of ≤2500 Da and is typically selected from the group consisting of:

[0190] Maytansines

[0191] Monomethyl auristatin

[0192] Calicheoamicins

[0193] Doxorubicins

[0194] ·Pyrrolobenzodiazepine (Pyrrolobenzodiazepine)

[0195] Methotrexate

[0196] Topoisomerase 1 inhibitors

[0197] Glucocorticoid Receptor Modulators (GRMs)

[0198] Taxanes

[0199] Anthracyclines

[0200] Alpha-amanitin, and / or

[0201] Cyclosporines.

[0202] Some examples include, but are not limited to, SN38, Exatecan, Dexamethasone, Budesonide, Mertansine, Ansamitocin, Ravtansin, DM4, DM1, Ozogamicin, Monomethyl Auristatin F (MMAF), and Monomethyl Auristatin E (MMAE).

[0203] Unlike immunotoxins and immunocytokines, effector molecules must be conjugated to the antibody in a separate step. Typically, this is achieved by using a linker / spacer consisting of p-aminobenzyl carbamate, which is conjugated to the free thiol group of a cysteine ​​residue within the antibody; a cathepsin-cleavable linker containing citrulline and valine; and an attachment group consisting of hexanoic acid and maleimide, to which the toxin is conjugated.

[0204] Here, the site specificity of the coupling reaction and the stoichiometry between the antibody and the toxin play an important role. One attempt to address these issues includes the so-called thiomab approach, in which antibodies are engineered with cysteine ​​residues to create a preferred coupling site for the linker / spacer (see Panowski et al., 2014, which is incorporated herein for implementation purposes).

[0205] Other attempts have utilized specific enzymes to couple toxins to antibodies in a stoichiometric and site-specific manner, such as sortases or transglutaminases (see, e.g., WO2014140317A1 and WO2020188061A1, the contents of which are incorporated herein for implementation purposes).

[0206] Antibody-radionuclide comprises at least one antibody or fragment thereof labeled with a radionuclide, such as Yttrium 90, Iodine 131 or Lutetium 177. Such molecules are disclosed, for example, in Steiner & Neri 2011, the contents of which are incorporated herein for practical purposes.

[0207] According to another aspect of the present invention, a pharmaceutical composition is provided, which comprises the antibody or fragment thereof described above, the nucleic acid described above, or the recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide described above, and optionally one or more pharmaceutically acceptable excipients.

[0208] According to another aspect of the present invention, a combination is provided, which comprises: (i) the antibody or fragment thereof described above, the nucleic acid described above, the recombinant immunotoxin described above, the immunocytokine, the antibody-drug conjugate or the antibody-radionuclide described above, or the pharmaceutical composition described above; and (ii) one or more therapeutically active compounds.

[0209] According to another aspect of the present invention, there is provided a use of the above-described antibody or fragment thereof, the above-described nucleic acid, the above-described recombinant immunotoxin, the above-described immunocytokine, the above-described antibody-drug conjugate or antibody-radionuclide, the above-described pharmaceutical composition or the above-described combination in the preparation of a drug for treating a human or animal subject. The human or animal subject

[0210] · Diagnosed with tumor disease,

[0211] Suffering from a tumor, or

[0212] At risk of developing a tumor;

[0213] Or for preventing the tumor disease.

[0214] This expression is considered to cover both Swiss-type claim language accepted in certain countries (in which case the brackets are deemed not to be present) and EPC2000 language (in which case the brackets and their contents are deemed not to be present).

[0215] According to another aspect of the present invention, a method for treating or preventing a tumor disease is provided, which comprises administering to a human or animal subject a therapeutically effective amount of the antibody or fragment thereof described above, the nucleic acid described above, the recombinant immunotoxin described above, the immunocytokine described above, the antibody-drug conjugate or antibody-radionuclide described above, the pharmaceutical composition described above, or the combination described above.

[0216] According to another aspect of the present invention, there is provided a treatment kit, the treatment kit comprising:

[0217] a) the above-described antibody or fragment thereof, the above-described nucleic acid, the above-described recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide, the above-described pharmaceutical composition or the above-described combination

[0218] b) a device for applying the composition, combination or combination, and

[0219] c) Instructions for use.

[0220] Example

[0221] Although the present invention has been described in detail by illustration and description in the drawings and the foregoing specification, such illustration and description should be regarded as illustrative or exemplary rather than restrictive; the present invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments may be understood and implemented by a person skilled in the art in practicing the claimed invention after studying the drawings, the disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The fact that certain measures are listed in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope.

[0222] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown from 5' to 3' end.

[0223] Materials and Methods

[0224] Genetic constructs of anti-CD79B antibodies

[0225] The full-length rituximab heavy chain (HC) and light chain (LC) sequences have been used to develop monoclonal antibody-based binder-toxin fusion proteins. The heavy chain and light chain variable region sequences of the anti-CD79B antibody were developed by the inventors through humanization of the murine antibody SN8 and fused with the human IgG1 Fc partial sequence.

[0226] Gene constructs containing anti-CD79B antibodies

[0227] Anti-CD79B antibodies were developed by hybrid humanization strategy of murine antibody (SN8). Full-length heavy and light chain antibody sequences have been used to develop antibody-based binder-toxin fusion proteins. Subsequently, the Anisoplin sequence was fused to the C-terminus of the heavy chain (HC) using the G4S sequence to obtain HC-G4S-Anisoplin+LC. The scFv-Fc, heavy chain and light chain portions were linked to Anisoplin or other ribotoxins with cleavage sites to achieve another binder-toxin fusion protein, thereby obtaining HC-FCS-Anisoplin+LC, HC-FCS-Anisoplin+LC, and LC-FCS-Anisoplin+HC-FCS-Anisoplin. These sequences were generated by gene synthesis with XbaI and IsceI restriction sites on both sides.

[0228] Transient expression in leaves of Nicotiana benthamiana plants

[0229] Nicotiana benthamiana was grown under a photoperiod of 16 hours light / 8 hours dark at 22 + / - 3°C. Leaves of 7-8 week old plants were transiently transformed by injection infiltration. Agrobacterium tumefaciens GV3101 (pMP90) containing an undisclosed plasmid containing the gene construct was cultured to its optical density at 600 nm (OD 600 ) reached about 0.8-1.0, and then the bacteria were collected by centrifugation at 3500 g for 10 minutes. Finally, the bacteria were adjusted to OD in infiltration buffer (10 mM MgCl2, 10 mM MES, 100 μM acetosyringone, pH 5.6). 600 The concentration of 0.5 was used and the mixture was infiltrated using a needleless syringe. The infiltrated area was harvested 4 and 6 days after infiltration. The whole leaf harvested 4 days after infiltration was used for Protein A purification.

[0230] Expression in tobacco (N. tabacum) cells

[0231] Tobacco (Nicotiana tabacum) plant suspension cells were cultured in plant medium for 5 days at 130 rpm and 25°C. The medium formulation was as described by Nagata et al. (1992), the contents of which are incorporated herein. Agrobacterium tumefaciens LBA4404 (pBBR1MCS-5.virGN54D) containing the pPZP-ATB binary plasmid was cultured to an optical density at 600 nm (OD 600) reaches about 0.8-1.0, then centrifuge for 5 minutes and collect bacteria at 2000g. Subsequently, plant cell and bacterial cell are co-cultivated in co-cultivation medium for 30 minutes, then centrifuge for 5 minutes at 2000g. After removing supernatant, cell is layered on solid co-cultivation medium and cultivated for two days. In the case of transient transformation, collect cell and wash three times, then cultivate in the plant culture medium containing cefotaxim (Cefotaxim) and carbenicillin (Carbeniclin), gather in the crops afterwards for further analysis. In the case of stable transformation, after two days of solid co-cultivation, wash cell and be layered on the plant culture medium containing selectivity kanamycin (kanamycin), cefotaxim and carbenicillin. Select callus after 4 weeks, and carry out subculture in solid culture medium or liquid suspension culture, for subsequent analysis.

[0232] Protein A purification

[0233] 4 days after agrobacterium infiltration, leaves were collected, weighed, and ground in a blender, using 2 mL of extraction buffer (0.1 M TRIS, 460 mM NaCl, 5 mM EDTA, 5 mM sodium pyrosulfite, pH 7.5) per gram of fresh infiltration leaves. Subsequently, the mixture was filtered through a double-layer Miracloth (Millipore). The filtrate was centrifuged at 40,000 g for 10 minutes at 4 ° C. The supernatant was loaded onto the protein A resin equilibrated with washing buffer in advance. The resin was then washed with 60 mM TRIS, 25 mM NaCl, 460 mM NaCl (pH 7.5) of 10 times of column volumes, and eluted with 100 mM glycine and 460 mM NaCl (pH 3.0), and the eluent was directly neutralized with 10% 1 M TRIS (pH 8.0). The protein fractions enriched were collected, dialyzed and frozen in liquid nitrogen.

[0234] The purified conjugate-toxin protein was visualized by SDS-PAGE.

[0235] Size Exclusion Chromatography

[0236] After protein A purification, the enriched protein fractions were loaded onto a Sephacryl S-300HR column (Cytivia). The main peak was collected and the residual peak was removed from the collected fractions before pooling.

[0237] Protein analysis: SDS-PAGE and Western blot

[0238] Proteins were boiled for 5 min in reducing or non-reducing SDS loading buffer (80 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue), centrifuged at 13,000 rpm for 5 min, and then separated by SDS-PAGE (4–20% polyacrylamide).

[0239] For Western blot, proteins were electrotransferred onto PVDF membranes (Biorad) using a semi-dry electrotransfer apparatus (Biorad Trans-Blot Turbo); subsequently, the membranes were blocked with 3% (w / v) skim milk powder in TBST buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.5) for 1 h at room temperature and then incubated with HRP-conjugated anti-human IgG Fc-specific region antibody (A0170; Sigma-Aldrich, diluted 1:10,000) or polyclonal primary antibody against Anisoplin (diluted 1:50,000, internal reference) in TBS-Tween 0.1% + 0.5% skim milk powder for 1 h at room temperature. Anti-human Fc antibody and anti-Anisoplin antibody were subsequently detected with HRP-conjugated anti-rabbit secondary antibody (Synabs, diluted 1:5000). Proteins were detected by enhanced chemiluminescence (Amersham Imager 600 / GE; GE Healthcare).

[0240] Protein Analysis: Analytical Size Exclusion Chromatography

[0241] Using Cytiva's high-resolution Superdex 200Increase 10 / 300GL column, Pure or The purified protein was analyzed by size exclusion chromatography on a Go chromatography system. The column was equilibrated with 2 column volumes of PBS at a flow rate of 1 mL / min. The purified protein samples with a concentration of 1 to 10 mg / mL were centrifuged at 20,000 g for 5 minutes. The supernatant was collected and 200 μL was loaded into a 100 μL capillary injection loop. Subsequently, the sample in the loop was loaded onto the column at a flow rate of 0.75 mL / min and eluted with 1.5 column volumes of PBS. The elution profile was monitored by ultraviolet absorption (UV).

[0242] In vitro cytotoxicity assay

[0243] The effect of the conjugate-toxin fusion protein on the viability of cell lines expressing CD79b was evaluated using Cell Titer Glo Assay (Promega, G9241). In this experiment, the single oxidation reaction of luciferin is catalyzed by luciferase in the presence of Mg2+ and ATP. The reaction produces a luminescent signal proportional to the number of living cells.

[0244] Depending on the cell line tested, cells were seeded in 96-well plates at a density of 2000 or 5000 cells / well, and 50 μL of growth medium (RPMI1640) was added to each well. 10 μL of conjugate-toxin fusion protein or buffer (PBS, 0.02% Tween) was added to 40 μL of growth medium to prepare serial dilutions of conjugate-toxin fusion protein. The mixture was added to the cells and incubated for 72 hours at 37°C, 5% CO2. The conjugate-toxin fusion protein was tested in duplicate wells. Buffer was used as a negative control, and the medium-only and cell-only groups were used as blank controls and untreated controls, respectively.

[0245] After 72 hours, the plate was equilibrated at room temperature for 30 minutes, and then 100 μL of CellTiter Glo reagent was added to each well. The plate was then placed on a shaker for 2 minutes, and then left to stand at room temperature in the dark for 10 minutes to allow the signal to stabilize. Finally, the luminescent signal was recorded.

[0246] To determine cell viability, the mean luminescent signal of the blank control (growth medium only) was subtracted from each well, and the mean luminescent signal of untreated cells was set to 100% viability. The mean signal of treated cells was then normalized and plotted as a function of the conjugate-toxin fusion protein concentration.

[0247] The anti-CD79b-based binder-toxin fusion protein was evaluated on a B-cell lymphoma cell line (B-NHL, CD79+) and non-target cells K562 (CD79-).

[0248] Immunohistochemistry-Tissue Microarrays (TMAs)

[0249] The conjugate-toxin fusion protein was labeled with fluorescein isothiocyanate (FITC) using the Thermo Scientific "Pierce" Antibody Labeling Kit.

[0250] Slides containing CD79b positive cell lines (B-NHL representative) and CD79b negative cells (K562) were used for method development and to determine the optimal concentration of the antibody for further studies. Cross-reactivity studies were performed using TMAs containing human normal frozen tissue (T6234701-1 / 2, Biochain Institute Incorp, CA, USA).

[0251] The fixation step was performed in cold acetone for 10 minutes, followed by air drying for 10 minutes. PBS + 10% normal human serum (Jackson Immunoresearch, 009-000-121) was added and incubated for 20 minutes. After removing the normal human serum, the tissue blocks were incubated with FITC-labeled conjugate-toxin fusion protein for 1 hour. After washing in PBS for 3 minutes, the tissue blocks were incubated with rabbit anti-FITC antibody (Serotec, #4510-7804) diluted 1:1000 for 30 minutes.

[0252] After washing in PBS for 3 minutes, the material was incubated with ready-to-use polymer BrightVision anti-rabbit / HRP (Immunologic, DPVR110HRP) for 30 minutes. The tissue blocks were washed in TRIS buffer (0.05M Trizmabase-Sigma, T1503-500g-dissolved in distilled water) for 3 minutes. The tissue blocks were incubated in DAB (50mg diaminobenzidine-Sigma, #D5637-5G-dissolved in 100ml TRIS buffer (see above) and 100ml H2O2-Merck, 1.07209.0250) for 5 minutes.

[0253] The tissue blocks were washed in distilled water for 3 minutes, then counterstained in hematoxylin for 10 seconds, followed by rapid washing with distilled water. The tissue blocks were dehydrated in 70%-90%-95% ethanol in sequence, 2 minutes per step, then dehydrated in 99.5% ethanol twice, 5 minutes each time, and finally dehydrated in X-tra solve (Medite, 41-5213-00) 3 times, 5 minutes each time. Sections were prepared with coverslips and mounting medium (Medite, 41-5219-00).

[0254] The staining results were judged as negative (0), weakly positive (1+), weakly to moderately positive (1-2+), moderately positive (2+), moderately to strongly positive (2-3+), or strongly positive (3+).

[0255] Cell line-derived xenografts

[0256] Representative B-NHL cell lines were injected into the peritoneal cavity of CB17.SCID mice. When the tumor size reached approximately 1 cm 3 When the average tumor volume reached the desired size, the animals were divided into 6 groups with 7 animals in each group and the average tumor volume was 0.139 cm 3 .

[0257] All conjugate-toxin fusion proteins used in vivo were purified by size exclusion chromatography (SEC) to remove aggregates. Animals were injected intravenously with a single dose of 20 mg / kg of ATB-747, ATB-580, ATB-693, ATB-697 and ATB-704 antibodies or vehicle (PBS, 0.02% Tween). Body weight and tumor volume were monitored three times per week.

[0258] The mean tumor volume for each group was plotted as a function of days after the start of treatment. Tumor growth curves were plotted for individual animals in each group.

[0259] For ethical reasons, when the tumor volume approaches 1.5 cm 3 The mice were killed.

[0260] Statistical analysis was performed using GraphPad Prism 9.3.1 software. To compare the different groups in the study, a two-way ANOVA was performed.

[0261] Cell viability assay

[0262] The cytotoxicity of purified conjugate-toxin fusion proteins has been evaluated on cancer cell lines. All humanized anti-CD79B toxin fusion proteins (ATB-580, ATB-693, ATB-697, ATB-704) showed damage to the activity of positive cell lines. In addition, we demonstrated that it is superior to Polivy (Polatuzumabvedotin), an ADC drug for CD79B on the market. These results reflect the superior mechanism of action of conjugate-toxin fusion proteins compared to antibody drug conjugates.

[0263] Off-target / cross-reactivity assessment

[0264] Figure 7 All humanized anti-CD79B toxin fusion proteins (ATB-580, ATB-693, ATB-697, ATB-704) showed no off-target binding. Only tissues containing targeted B cells (lymph nodes, spleen, thymus) were stained. Similar data were obtained on frozen tissues of cynomolgus monkeys and mice (data not shown).

[0265] In vivo efficacy

[0266] Polatuzumab

[0267] like Figure 8As shown in the results, all humanized recombinant anti-CD79B immunotoxins (ATB-580, ATB-693, ATB-697, ATB-704) showed potent and durable anti-tumor effects in the CDX model. In particular, ATB-580 outperformed the benchmark drug polatuzumab recombinant immunotoxin ATB-747 (fused to the protein toxin anisoplin via a G4S linker) ( Figure 8 ).

[0268] Analytical Size Exclusion Chromatography (SEC)

[0269] Using Cytiva's high-resolution Superdex 200Increase 10 / 300GL column, Pure or The purified protein was analyzed by size exclusion chromatography on a Go chromatography system. The column was equilibrated with 2 column volumes of PBS at a flow rate of 1 mL / min. The purified protein samples with a concentration of 1 to 10 mg / mL were centrifuged at 20,000 g for 5 minutes. The supernatant was collected and 200 μL was loaded into a 100 μL capillary injection loop. Subsequently, the sample in the loop was loaded onto the column at a flow rate of 0.75 mL / min and eluted with 1.5 column volumes of PBS. The elution profile was monitored by ultraviolet absorption (UV).

[0270] Analytical SEC showed that the humanized anti-CD79B toxin fusion protein had a low tendency to aggregate, with aggregation rates ranging from 3% (ATB-704) to 8% (ATB-508) ( Figure 4 The inventors observed that compared with the benchmark drug Polatuzumab ( Figure 5 ), the aggregation tendency of the humanized anti-CD79B toxin fusion protein was significantly reduced compared to ATB-452, while the aggregation rate of Polatuzumab was 13% (ATB-452).

[0271] References

[0272] · G.&Milstein,C.(1975):Continuous cultures of fusedcellssecreting antibody of predefined specificity.In:Nature.Bd.256,S.495–497.Jonsson and Malmquist,Advances in Biosnsors,2:291-336(1992)

[0273] ·Plückthun and Skerra,Meth.Enzymol.,178:497-515(1989)and in Day,E.D.,Advanced Immunochemistry,Second Ed.,Wiley-Liss,Inc.,New York,N.Y.(1990)

[0274] ·Harding,The immunogenicity of humanized and fully humanantibodies.MAbs.2010May-Jun;2(3):256–265.

[0275] ·Eylenstein,et al,Molecular basis of in vitro affinity maturationandfunctional evolution of a neutralizing anti-human GM-CSF antibody,mAbs,8:1,176-186(2016)

[0276] ·Kabat et al.,J.Biol.Chem.252,6609-6616(1977)and Kabat et al.,Sequences of protein of immunological interest.(1991)

[0277] ·Chothia et al.,J.Mol.Biol.196:901-917(1987)

[0278] ·Brühl H,Cihak J,Talke Y,Rodriguez Gomez M,Hermann F,Goebel N,RennerK,Plachy J,Stangassinger M,Aschermann S,Nimmerjahn F,Mack M.B-cell inhibitionby cross-linking CD79b is superior to B-celldepletion with anti-CD20antibodies in treating murine collagen-inducedarthritis.Eur JImmunol.2015Mar;45(3):705-15.

[0279] ·Tilly H et al.Polatuzumab Vedotin in Previously Untreated DiffuseLargeB-Cell Lymphoma.N Engl J Med 2022;386:351-363

[0280] ·Okazaki et al., Blood, 81:84-94 (1993)

[0281] ·Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014 Jan-Feb; 6(1): 34-45. doi: 10.4161 / mabs.27022. PMID: 24423619; PMCID: PMC3929453.

[0282] ·Steiner M, Neri D. Antibody-radionuclide conjugates for cancer therapy: historical considerations and new trends. Clin Cancer Res. 2011Oct15; 17(20):6406-16

[0283] sequence

[0284] The following sequences form part of the disclosure of this application. This application also provides an electronic sequence listing that complies with the WIPOST 26 standard. To avoid ambiguity, if there is a difference between the sequence in the table below and the electronic sequence listing, the sequence in the table below should be considered the correct sequence. In some cases, a signal peptide may be included in the copied sequence. In this case, the sequence should be considered as public content regardless of whether it contains or not a signal peptide. A convenient tool for identifying signal peptides in a given protein sequence is SignalP-6.0 provided by the Technical University of Denmark, which can be accessed through the following link:

[0285] https: / / services.healthtech.dtu.dk / service.php?SignalP.

[0286] It is also noted that in certain embodiments, the corresponding amino acid sequence of the toxin shows a deimmunized version thereof. All embodiments should be considered to disclose the wild-type toxin sequence or a deimmunized variant thereof.

[0287] Table 4: Sequence

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

Claims

1. An antibody or a target binding fragment or derivative thereof that retains the ability to bind to human CD79b, a) the antibody or target binding fragment or derivative thereof comprises a set of six heavy chain / light chain complementarity determining regions (CDRs), the heavy chain / light chain complementarity determining regions being contained in a heavy chain / light chain variable region sequence pair selected from any one of the following SEQ ID NO pairs: SEQ ID NO: 3 and 4, SEQ ID NO: 13 and 14, SEQ ID NO: 23 and 24, or SEQ ID NO: 33 and 34, b) the antibody or target binding fragment or derivative thereof comprises a set of six heavy chain / light chain complementary determining regions (CDRs), the heavy chain / light chain complementary determining regions being arranged in the order of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 and selected from: i) SEQ ID NO: 5, 6, 7, 8, 9 and 10, ii) SEQ ID NO: 15, 16, 17, 18, 19 and 20, iii) SEQ ID NO: 25, 26, 27, 28, 29 and 30, or iv) SEQ ID NO: 35, 36, 37, 38, 39 and 40, c) the antibody or target binding fragment or derivative thereof comprises a set of heavy chain / light chain complementarity determining regions (CDRs) as described in option b), and at least one of the CDRs has a maximum of 3 amino acid substitutions relative to the CDR contained in the corresponding SEQ ID NO, and / or d) the antibody or target binding fragment or derivative thereof comprises a set of heavy chain / light chain complementarity determining regions (CDRs) as described in option b) or c), and at least one of the CDRs has a sequence identity of ≥66% with the CDR contained in the corresponding SEQ ID NO. in, The CDRs are embedded in a suitable protein framework so as to be able to bind to human CD79b.

2. The antibody or fragment of claim 1, wherein the antibody is a humanized antibody or fragment.

3. The antibody or fragment according to any one of claims 1 to 2, comprising a) a heavy chain / light chain variable region (HCVD / LCVD) pair listed in any of the following SEQ ID NO pairs: SEQ ID NO: 3 and 4, SEQ ID NO: 13 and 14, SEQ ID NO: 23 and 24, or SEQ ID NO: 33 and 34, b) a heavy chain / light chain variable region (HCVD / LCVD) pair as described in a), and satisfying The heavy chain variable region (HCVD) has ≥ 80% sequence identity with the HCVD contained in the corresponding SEQ ID NO, and / or a light chain variable region (LCVD) having ≥ 80% sequence identity with the LCVD contained in the corresponding SEQ ID NO, c) a heavy chain / light chain variable region (HCVD / LCVD) pair as described in option a) or b), wherein at least one of said HCVD or LCVD has a maximum of 10 amino acid substitutions relative to the HCVD or LCVD comprised in the corresponding SEQ ID NO, The antibody or fragment is capable of binding to human CD79b.

4. The antibody or fragment of any one of claims 1 to 3, wherein at least one amino acid substitution is a conservative amino acid substitution.

5. The antibody or fragment of any of the preceding claims, wherein the human CD79b to which the antibody or fragment binds comprises: a) the amino acid sequence shown in SEQ ID NO: 41-44, or b) an amino acid sequence having at least 80% sequence identity to SEQ ID NOs: 41-44.

6. The antibody or fragment according to any one of the preceding claims, which is a monoclonal antibody, or a target binding fragment or derivative thereof that retains the ability to bind to the human CD79b target.

7. The antibody or fragment according to any one of the preceding claims, which is at least one selected from the group consisting of: IgG, scFv, Fab or (Fab)2.

8. A nucleic acid encoding at least one chain of the antibody or fragment as claimed in any one of the preceding claims.

9. A recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide conjugate comprising the antibody or fragment as claimed in any one of the preceding claims.

10. A pharmaceutical composition comprising the antibody or fragment as described in any one of claims 1 to 7, the nucleic acid as described in claim 8, or the recombinant immunotoxin, immunocytokine, antibody drug conjugate or antibody-radionuclide conjugate as described in claim 9, and optionally one or more pharmaceutically acceptable excipients.

11. A combination comprising: (i) an antibody or fragment as described in any one of claims 1 to 7, a nucleic acid as described in claim 8, a recombinant immunotoxin, an immunocytokine, an antibody-drug conjugate or an antibody-radionuclide conjugate as described in claim 9, or a pharmaceutical composition as described in claim 10; and (ii) one or more therapeutically active compounds.

12. Use of the antibody or fragment according to any one of claims 1 to 7, the nucleic acid according to claim 8, the recombinant immunotoxin, immunocytokine, antibody-drug conjugate or antibody-radionuclide conjugate according to claim 9, the pharmaceutical composition according to claim 10 or the combination according to claim 11 in the preparation of a medicament for treating a human or animal subject, wherein the human or animal subject · Diagnosed with tumor disease, Suffering from a tumor, or At risk of developing tumors, Or for preventing the tumor disease.

13. A method for treating or preventing a tumor disease, the method comprising administering to a human or animal subject a therapeutically effective amount of an antibody or fragment as described in any one of claims 1 to 7, a nucleic acid as described in claim 8, a recombinant immunotoxin, an immunocytokine, an antibody-drug conjugate or an antibody-radionuclide conjugate as described in claim 9, a pharmaceutical composition as described in claim 10, or a combination as described in claim 11.

14. A treatment kit comprising: a) an antibody or fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, a recombinant immunotoxin, an immunocytokine, an antibody-drug conjugate or an antibody-radionuclide conjugate according to claim 9, a pharmaceutical composition according to claim 10 or a combination according to claim 11, b) a device for administering said composition, combination or combination, and c) Instructions for use.

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